Abstracts of papers presented at the 8th conference of the Entomological Society of Israel Abstracts of papers presented at the 17th congress of the Israeli Phytopathological Society

M E E T I N G S

]"ILll'71FJ1D] N ~

ABSTRACTS OF PAPERS PRESENTED AT

THE 8TH CONFERENCE OF THE E N T O M O L O G I C A L S O C I E T Y OF I S R A E L

Basic and Applied Research on Fruit Flies (Diptera: Tephritidae) of Agricultural Importance in Israel

January 18, 1996 ARO, The Volcani Center, Bet Dagan, Israel

The Israeli Tephritid Fauna Revisited

Amnon Freidberg Dept. of Zoology, The George S. Wise Faculty of Life Sciences, Tel-Aviv University,

Tel Aviv 69978, Israel [Fax: +972-3-6409403]

Forty-five genera and 91 species of Tephritidae (Diptera) are currently recorded from Israel. These numbers constitute an addition of three genera and six species over the last published faunal treatment (Freidberg and Kugler, 1989, Fauna Palestina, Insecta IV - Diptera: Tephritidae). In addition to the two well known fruit pests in Israel, the Mediterranean fruit fly (Ceratitis capitata [Wiedemann]) and the olive fruit fly (Bactrocera oleae (=Dacus oleae) [Gmelin]), other, new or little known pests were commented on: (i) Myiopardalis pardalina, the 'melon fly' of Judea and Samaria, which infests melon and snake cucumber; (ii) Euleia heracleii (Linnaeus), the celery fly, is a leafminer in various Apiaceae, including celery in Europe and Smyrnium olusatrum L. in Israel; (iii) Capparimyia savastani (Martelli), the caper fly, infests flower buds of at least two species of Capparis, including C. spinosa L., which is grown commercially in some Mediterranean countries;

Phytoparasitica 24: 2, 1996 127

(iv) Carpomya incompleta (Becker), infesting fruit of all Zizyphus species in Israel, including the introduced Z. mauritiana; (v) Acanthiophilus helianthi (Rossi), Chaetorellia carthami Stackelberg and a few other, less important species, are pests in flowerheads of safflower (Carthamus tinctorius L.); and (vi) Terelliafuscicornis (Loew), the artichoke fly, infesting the flowerheads of cultivated and wild artichoke. Recent interceptions of new pests in Israel were reported, and a warning was given against the possible introduction into Israel of additional pests of this family.

The Mediterranean Fruit Fly in Israel: Status, Control, Conflicts and Prospects

Yoram R6ssler The Israel Cohen Institute for Biological Control, Citrus Marketing Board of Israel,

Bet Dagan 50250, Israel [Fax: +972-3-9683838]

The Mediterranean fruit fly (medfly) is a major pest of almost all fruit crops in Israel (citrus, deciduous fruits, table grapes and subtropical fruits). It is a quarantine pest in many countries which ban totally or restrict partially the importation of flesh produce (not only fruit) from Israel. The impact of the pest exceeds, therefore, the actual crop loss. Control measures in Israel, as in many other medfly countries, rely heavily on chemical applications, either as ground or aerial sprays. Medfly control in citrus and deciduous orchards is carried out centrally and under a special Ministry of Agriculture regulation. The country-wide program has been based, since 1957, on a network of monitoring traps containing a male attractant, located in commercial orchards, and inspected weekly. Presence of flies in these traps triggers the initiation of aerial application of malathion and protein bait sprays. The method remained basically unchanged since 1957 and proved to be cost effective and efficient. However, recent shifts in the human population structure, a change in the public awareness regarding the massive use of insecticides, regulations concerned with environmental quality, and the detrimental effects on the fauna of beneficial insects in some crops, have cast doubts on the overall validity of the current control method. Unfortunately, there is yet no viable and economically acceptable alternative to aerial medfly control. Scientists throughout the world are searching for new solutions to the medfly problem. In Israel the medfly, although a major pest, has not received its rightful place among the scientific community. Recent interest shown by young scientists of various disciplines, in basic and practical research on that pest, is a refreshing change in attitude, and will, it is hoped, bear the expected fruits. These scientists are urged to bear in mind that, although the accumulation of basic knowledge is important and valid, their responsibility to the community is to offer new, environmentally acceptable, economically feasible and safer methods of managing the medfly, and to keep the pest under acceptable control levels.

Population Genetics of the Mediterranean Fruit Fly in Israel

David Nestel and Smadar Hirsh Dept. of Entomology, ARO, The Volcani Center, Bet Dagan 50250, Israel [Fax: 972-3-9604180]

We are currently utilizing the Polymerase Chain Reaction-Random Amplified Polymorphic DNA (PCR-RAPD) technique to investigate the population genetics of the Mediterranean fruit fly (medfly) in Israel. In this procedure, short stretches of DNA are amplified in a polymerase chain reaction directed by an oligonucleotide of arbitrary sequence. The amplification results in a characteristic set of products that can be used as a polymorphic genetic marker. Resulting patterns can be used to calculate (by means of statistical methods, i.e., multivariate classification methods) the within- population genetic variability and the genetic distance between populations. Preliminary results suggest that the within-population variability of the medfly is high, and also that different populations

128 Meetings

of the medfly in the country can be differentiated by their distinct genetic patterns. It is expected that the study of the genetic patterns of the different populations of the medfly in the country will allow us to understand the colonization and dispersal history of the fly in Israel, and the effect of geographic isolation and breeding host on the genetic makeup of the population.

Information derived from this study is expected to complement our knowledge on the ecology of the medfly in Israel. The information may be used in the future design of alternative strategies of control, and in the application of the sterile insect technique in Israel.

Relationship between Temporal and Spatial Distribution of Mediterranean Fruit Fly

Populations and Host Phenology

Nimrod Israely, 1 Boaz Yuval 1 and David Nestel 2 1Dept. of Entomology, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100 [Fax: +972-8-9466768]; and 2Dept. of Entomology, ARO, The Volcani Center, Bet Dagan, Israel

Little is known about the spatial and temporal distribution of Mediterranean fruit fly (Ceratitis capitata) populations, and to what extent it is related to host phenology and host spatial distribution. To address this question we monitored, with trimedlure traps, medfly populations during two seasons of activity, by intensively sampling a 6 km 2 area in the surroundings of Kibbutz Zova (in the Judean Hills). Our findings suggest that three main factors ('predominant environmental factors') seem to govern the spatial and temporal distribution of the Mediterranean fruit fly: extreme temperatures, host range and host spatial distribution. The population of the adult Mediterranean fruit fly decreased to zero levels during the winter months and remained such until spring, when early flies were again captured in the traps. Thereafter, the spatial distribution and buildup of the population during summer and autumn were closely associated with the phenology of the different fruit hosts, and with their spatial distribution. Additional factors that determine the spatial distribution of the fly population, and that affect the degree of influence of the predominant environmental factors, are: insecticide applications, density of host fruits and distance between hosts.

Daily Activity Pattern of the Mediterranean Fruit Fly in the Coastal Plain of Israel

Meirav S. Warburg and Boaz Yuval Dept. of Entomology, The HebreW University of Jerusalem, Faculty of Agriculture,

Rehovot 76100, Israel [Fax: +972-8-9466768]

Daily observations of individual Mediterranean fruit flies (Ceratitis capitata [Weidemann]) were conducted at two sites in the coastal plain of Israel during two summer seasons. One of the sites was a mixed orchard of guava (Psidium guajava) and pitanga (Eugenia uniflora) and the other an apple grove adjacent to a fig grove. On a total of 25 observation days we captured 885 flies engaged in different activities.

Common activity patterns were found at both sites. Flies of both sexes began their day resting on leaves in the sun, and fed briefly as temperatures rose. Females then dedicated their time mainly to ovipositing, but they also fed and mated. Males participated in leks on the foliage of specific trees. This was almost their exclusive activity until the late afternoon hours when lekking ceased and an alternative strategy of fruit guarding was employed by some males. Evening was the primary feeding time for both sexes. Over-ripe and rotten fruits were preferred substrates. Both sexes were also found feeding on the surface of leaves and fruit. Females appear to compensate for lack of protein in their sugar-rich diet by consuming bi~ d droppings.

Phytoparasitica 24:2, 1996 129

While male activities were regimented and discrete, females were engaged in different activities in an opportunistic manner throughout the day. Different activities were related to certain hosts and specific locations on the host. In particular, lek sites appear to depend on specific host parameters. During lekking hours all males are concentrated at such sites, and cannot be found elsewhere in the habitat. The synchronized behavior of the population indicates a daily periodic migration among reproduction, oviposition, feeding and resting sites.

Male--Female Interactions in the Mediterranean Fruit Fly: Sperm Transfer and Storage

Boaz Yuval, Sigal Blay and Roy Kaspi Dept. of Entomology, The Hebrew University of Jerusalem, Faculty of Agriculture,


Several aspects of sperm transfer and storage in the Mediterranean fruit fly, Ceratitis capitata (Wiedemann), were examined. We determined how many sperm are available to virgin males, how many sperm they ejaculate, and how many are subsequently stored by females. In addition we established how sperm are allocated between the two spermathecae and investigated sperm load and sperm allocation patterns in females captured in the field in Israel. Male testes contained on the average 34,300 (:t:2,500) sperm cells before copulation and 14,759 (4-1,400) after copulation. It is intriguing that most of this ejaculate never reaches the spermathecae. Spermathecae of once- mated laboratory-reared females contained 3,2 l 2 (4-212) sperm, similar to the amount found in field- collected females, 3,612 (-t-237). We suggest that the excess sperm is used by females as a nutritional gift. Paradoxically, male nutritional stress significantly elevated the number of sperm cells produced by the males and the number stored by females.

Continuous access to males in the laboratory resulted in significantly elevated numbers of stored sperm cells (on the average 5,250 +790), indicating that multiple mating is common. The abundance of sperm stored by field-collected females supports this conclusion. Sperm allocation between the spermathecae, in both field and laboratory females, is significantly non-random; one organ (either left or right) contains significantly more sperm than expected by chance, suggesting that allocation of sperm is controlled, either by the male or by the female.

Factors Affecting the Reproductive Success of Male Mediterranean Fruit Flies

Sigal Blay and Boaz Yuval Dept. of Entomology, The Hebrew University of Jerusalem, Faculty of Agriculture,


Success of medfly control by means of the sterile insect technique depends largely on the competitive ability of released males. The objective of this study was to examine the relationship between a male's size and nutritional status on the one hand, and his ability to acquire mates and the subsequent receptivity of these females on the other hand. It was found that diet has a significant effect on the speed with which males are selected by virgin females. Protein-fed males began to copulate significantly faster than sucrose-fed males (KSa=I.5; n=i24; P=0.02). Size was also significantly correlated with mate acquisition, particularly in males fed on sugar (F=I 9.15; n= 124; P<0.001).

Remating on the day following the first copulation was high: 61% of females (n=135) mated to protein-fed males and 76% of females (n=100) mated to sugar-fed males (X 2 = 5.4; P=0.02). The latter females tended to remate sooner than the females mated to protein-fed males, and were

130 Meetings

choosier in regard to the size of their second mate (F=10.5; P=0.O018). We conclude that the size and nutritional status of a male significantly affect his reproductive success, both by gaining copulation with a virgin female and subsequently by postponing the female's next copulation.

How to Improve the Eradication of the Mediterranean Fruit Fly with the Use of Mass-Reared Flies

Oren Hasson Dept. of Entomology, The Hebrew University of Jerusalem, Faculty of Agriculture,


The sterile insect technique (SIT) is widely used for the eradication of the Mediterranean fruit fly (Ceratitis capitata). Nevertheless, experiments have shown that the mating success of the sterile males is extremely poor, and that they mate at frequencies far lower than expected by random mating alone. This is very likely the result of natural selection that is operating in the mass-rearing regime. The flies are raised at very high densities, their diurnal activity pattern changes, they are less active than wild flies, their generation turnover increases dramatically and, accordingly, they mature early. As a result, it is very likely that their mating strategies will change as well: they cannot lek as they do in nature, and it is quite probable that the mating strategies of males and females evolved by natural selection to be different. In addition, fluctuation in larval densities, and perhaps also in food quality, appears to have a dramatic impact on the male's potential to mate with females, via changes in the male's morphology. All these factors should be carefully studied in order to improve the effectiveness of SIT in achieving medfly eradication.

Trimedlure Efficacy for Trapping Mediterranean Fruit Flies in an Organic Citrus Orchard

Amos Rubin, 1 Arieh Landesman, 2 Edva Zakkay, 2 Ruthi Levy 2 and Salo Meidan 2 1 Consulting Service for Biological and Integrated Control, Givat Shemu'el 51905 (Present address:

Ministry of Agriculture, Plant Protection and Inspection Services, Bet Dagan 50250 [Fax: +972-3-9681507]); and 2Kibbutz Gan Shemu'e138810, Israel

Rapid drying up of common Mediterranean fruit fly (Ceratitis capitata [Wiedemann]) traps in organic citrus orchards, led us to try a different kind of trap (prepared by the Adhestick Factory, Ramla, Israel). Aerosol containers of 300 ml were stuffed with 10% trimedlure pheromone and glue; the same without trimedlure served as control. This material was sprayed on a plastic substrate. The experiment took place at Kibbutz Gan Shemu'el in an organic 34-year-old grapefruit orchard (1959 plantation). The kibbutz factory markets in Europe the organic juice of this citrus orchard. It is thus necessary to provide juice both free of pests and without use of any pesticide.

The number of flies that were trapped on a trimedulure trap (size 60 x 40 cm) was 46.5 vs 1.3 in the control trap. 306 flies were trapped in 6.6 trimedlure traps placed in 0.1 ha, vs 9 flies in the control traps. At a trap size of 10 x 10 cm, altogether 106 flies were captured per 0.1 ha (0 in the control traps), and with the 20 x 40 cm size traps, 132 flies were trapped per 0.1 ha vs 7 flies in the control traps. In a second experiment traps were placed, at distances of 3, 5 and 8 trees from one another. Altogether 184 flies were trapped per 0.1 ha on the trimedlure traps, when the distance between the traps was 3 trees, vs 4 flies in the control traps. It is possible that wind streams carried flies to the external traps in the row. Therefore in a calculation that omitted the external trap, 153 flies were trapped per 0.1 ha in the trimedlure traps, when the distance between the traps was 3 trees; 9 flies were trapped per 0.1 ha in the control traps, when the distance between the traps was 5 trees.

It is concluded that the closer the placement of the traps, the more flies are caught per 0.1 ha. This system is cheap and, in comparison with the old traps that dried up quickly, convenient and easy to operate, The results are satisfactory, and the pest is not attacking the organically grown grapefruits in the citrus orchard. Phytoparasitica 24:2, 1996 131

Development of a Food Bait for the Mediterranean Fruit Fly

Michal Mazor Dept. of Entomology, ARO, The Volcani Center,


Reports on the use of food lures attractive to fruit flies as baits (combined with poison or in traps) have been published since the beginning of the century. The first commercial fruit fly bait (PIB-7), marketed in the 1950s, was a corn protein hydrolysate developed on the basis of McPhail's findings on the attraction of fruit flies to protein hydrolysates. Further commercial products have been developed since then, but there has been no breakthrough that improved their efficiency. Numerous studies were conducted to identify attractive volatiles released from a commercial protein hydrolysate and by the biological degradation of nutrients from protein hydrolysates. However, the results of these studies were solely two new bait mixtures without improved attractancy.

Ammonia and some ammonia-releasing compounds were tested, among other substances, as baits for fruit flies, but there are conflicting results concerning their effectiveness. While testing the relationship between increasing the concentrations of ammoniacal aqueous solutions and the positive response of the flies, we found a clear correlation between the two parameters, which is linear up to the concentration at which the ammonia solution turns rejective. This may be the reason for contradictory results between different studies in which the release rate of ammonia was not measured.

Feces are a common source for proteinaceous food in nature, and are attractive for fruit flies. In the search for alternative substances which might render the bait more attractive, we examined a series of poultry and dairy manures. Prelimiminary results were promising, insofar as the manures tested were as attractive as, and in a few cases even more attractive than, the commercial baits Naziman (Tamogan, an Israeli product), Buminal (Bayer, Leverkusen, Germany) and Nu-Lure (the new name for PIB-7).

Data from the literature and our findings support our conjecture that ammonia is the key factor in the attractiveness of a protein hydrolysate bait. For example, by raising the pH of the bait solution, more ammonia is released and the solution becomes more attractive.

The main aim of this research is to develop a more efficient bait and to avoid the most prominent disadvantage of the conventional baits, namely, batch-to-batch variability. We hope to overcome this by composing the bait from defined chemicals. The development of the bait is a two-pronged effort: (i) locating a substance with a constant and optimal release rate of ammonia for attracting the flies; and (ii) composing a combination of the above substances with phagostimulatory and arrestant materials, to encourage the flies to make contact with the bait and consume it.

The Economic Damage Caused by the Olive Fruit Fly, Bactrocera oleae*

Fathi Ab d el-Hadi Ministry of Agriculture, Afula 18120, Israel [Fax: +972-6-528205]

In the Mediterranean basin, which has 98% of the world's cultivated olive trees, the olive tree is of significant socio-economic importance. These trees number about 800 million and occupy a surface area of approximately 10 million ha. They produce about 1.8 million metric tons per annum of olive oil, in addition to 800,000 metric tons of table olives. The losses which insect pests, fungi

* Lecture not presented at the Conference.

132 Meetings

and weeds cause to this crop are approximately 30% of the production. The damage caused to harvested fruits by insect pests is at least 15% of production, which equates to $US 800 million per annum. This occurs despite the fact that olive growers spend annually more than $US 100 million combating these pests.

The olive fruit fly (Bactrocera oleae (=Dacus oleae) [Gmelin]), which causes the greatest losses to the olive yield, has a variable number of generations per annum, depending upon the geographic area. Climatic factors, especially temperature, have a great influence on the insect biology. At less than 12~ female reproductivity comes to a halt and at below 9~ incubation of the eggs, larva growth and nymph development cease. At temperatures exceeding 30~ the female no longer lays eggs.

The main means of insect pest control has been through the use of conventional pesticides. However, pesticide residues have been detected in olive oil and in the environment, causing concern in most olive-growing countries and leading to a concerted effort to reduce the amount of pesticides applied.

Various control methods are in use in Israel and elsewhere: 1. Preventive treatment: Hydrolysate as an attractant in conjunction with an insecticide; aerial and ground sprays. 2. Curative treatments: Cover sprays with diethoate every 3 weeks, or according to adult catches. 3. Integrated pest management: Use of pheromones.

Three main strategies involving semiochemicals have been pursued in the development of IPM strategies for olive pests (mass trapping, lure and kill, and mating disruption).

During the last 20 years notable progress has been achieved in our knowledge of the chemical ecology of olive pests such as the olive fly and the olive moth. Several semiochemicals have been isolated, identified and synthesized from these two insects and used to monitor their populations. A sex pheromone released by virgin females attracts male B. oleae. The principal component of this sex pheromone was identified in late 1979 as 1,7-dioxaspiro [5,5] undecane. Other components were identified from the sex pheromone by various workers, but none was found in subsequent field trapping experiments to be critical in attracting male B. oleae. Many projects throughout the world use the IPM strategy for controlling olive pests. The most well known is the one in Ja6n, Spain, where there are approximately 600,000 ha of olives, 250,000 ha of which are included in the project.


MEETINGS

S

ABSTRACTS OF PAPERS PRESENTED AT ~ /

T H E 17TH CONGRESS OF q"t'7"OPATt40~'O T H E ISRAELI P H Y T O P A T H O L O G I C A L SOCIETY

February 19-20, 1996

ARO, The Volcani Center, Bet Dagan, Israel

Opening Lecture

Genetic Engineering of Crop Plants: Advantages v s Risks

Y. Gafni Dept. of Plant Genetics, ARO, The Volcani Center, Bet Dagan 50250, Israel

[Fax." +972-3-9669642]

It has been more than l0 years since scientists all over the world began using genetic engineering to develop transgenic plants. To date, there have been thousands of different field tests with transgenic plants. In many cases genes of potentially agricultural benefit were introduced. In most of the cases, genes that are involved in herbicide or insecticide resistance were used for the transformation. Among these, the best known case is the introduction of the insecticidal endotoxin from Bacillus thuringiensis into crop plants.

Today, tremendous effort is invested in rendering plants resistant to fungi, nematodes and viruses; in several cases the results are very promising. In various trials to improve the quality of fruits and vegetables, several different approaches were taken and in one case the transgenic tomatoes produced became known for their quality and extended shelf life and are now being sold as 'Flavar Savar' in supermarkets in the USA.

Genetic engineering is also a powerful tool in the production of hybrid seeds as well as of plants having proteins with high therapeutic value.

Transgenic crops represent a great promise for a better future. However, are there also risks involved in using these crops? One should always bear in mind that crop plants are defined as such by us and can transfer traits by pollination to wild plants, sometimes to weeds. If resistance to herbicides will be transferred to weeds, agriculture will have to bear the heavy costs. Farmers will be obliged to use new herbicides and in increased quantities. Another concern is using genes for resistance to antibiotics in the process of selecting transgenic plants in the laboratory. This is a point that should be considered carefully. Although we believe these genes can not be acquired by bacteria in our digestive systems, we still must be aware of the public's fear and try to overcome it. The same holds true for using genes coding for harmless antisense-RNA, to block biosynthetic pathways in the plant. We know it is all fight to use it but the consumer has to be convinced of that, too. It is our duty not


just to make the 'genetic engineering revolution' a better reality, but also to explain its advantages to all. If we succeed, we will have new ways of protecting crops with a better future for our agriculture.

A: NEW AND RECURRING BACTERIAL AND FUNGAL DISEASES

Detection of Brown Rot in Imported Potato Seeds

Leah Tsror (Lahkim), 1 0 r l y Erlich, 1 C. Chen, 2 I. Ben-Ze'ev, 2 G. Kritzman 3 and D. Zutra 3 1Dept. of Plant Pathology, ARO, Gilat Regional Experiment Station, M.P. Negev 2, 85410

[Fax: +972-7-926337]; 2plant Protection and Inspection Services, Ministry of Agriculture, Bet Dagan 50250; and aDept, of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel

Brown rot in potatoes is a quarantine disease caused by Pseudomonas solanacearum. This systemic disease causes wilting, growth retardation, stunting, chlorosis of leaves, browning of vascular bundles of tubers and secretion of bacterial exudate from eyes and stolons. There are five known races of this soilborne pathogen, which are differentiated by their host range. Although the disease is endemic to tropical and subtropical zones, race 3 has been reported to overwinter and spread in areas north of latitude 59~ posing a threat to potato production in Europe. Sources of infection are seed tubers, plant residues, and infected soil.

Although Israel was officially notified by Holland that there was an outbreak of the disease in one area of seed potato production, it was decided to continue importing seed tubers from Holland (10,000 tons). However, Israeli authorities required a health certificate, based on laboratory tests. In addition, it was decided to perform a laboratory examination of samples from all arriving seed lots. Samples of 200 tubers from each 25-ton batch are taken by inspectors of the Plant Protection and Inspection Services for laboratory testing. The detection of P. solanacearum is based on extraction of the bacteria from the vascular bundle of the tubers, and plating onto a semi-selective medium. Following incubation at 30~ for 3 days, characteristic colonies of the virulent type are transferred to a growth medium for 24 h. Identification is accomplished by immunological methods, such as agglutination, ELISA reaction and immunofluorescent staining, using specific antibodies prepared against virulent isolates obtained originally from potatoes and by fatty acid analysis by gas chromatography. Pathogenicity of suspected isolates is tested by inoculation of tomato and potato plants. Of the more than 400 samples tested so far, only one lot of seeds is suspected as being infected. Infected lots must be destroyed or returned to the country of origin.

P solanacearum is perceived as so grave a danger, that once a field is discovered to be infected it must lie fallow (or be used to grow only wheat) for a period of 5 years. It is imperative to prevent the pathogen's invasion into Israel, since it would have serious consequences for the export of agricultural products. (L)*

The Watermelon Fruit Blotch Disease and Other Diseases Caused by Acidovorax avenae

I. Assouline Spectrolab (Micro 1995), Rehovot 76120, Israel [Fax: +972-8-9361247]

The watermelon fruit blotch disease (WFB) is caused by a rod-shaped, non-fluorescent Gram negative bacterium, with one polar flagellum. An outbreak of the WFB disease in commercial fields was reported from Florida in 1989. Differences in glucose uptake, hypersensitivity reaction (HR) symptoms on tobacco leaves and development of symptoms on watermelon cotyledons and fruit are responsible for the different names given to this bacterium: Pseudomonas pseudoalcaligenes subsp.

*L = lecture sessions; P = poster (market place) sessions.

136 Meetings

citrulli and, recently, Acidovorax avenae subsp, citrulli; the latter was demonstrated to be seed- transmissible.

A similar bacterium was intercepted for the first time in Israel during March 1992 and then in spring 1993 on watermelon seedlings grown from imported seed in a greenhouse. The disease was eradicated in the greenhouse and was not found in the field. The isolate recovered from the diseased seedlings was unable to use glucose as a sole carbon source and was HR-negative on tobacco leaves. The pathogen was identified as Acidovorax avenae by morphological characters, biochemical tests and its fatty acids profile. During May 1995 diseased eggplant seedlings, grown from imported seed, were observed in a greenhouse. The symptoms were small necrotic leaf spots, with or without a chlorotic halo. The causal agent was again identified as A. avenae by morphological and biochemical characters and by its fatty acids profile; the disease in the greenhouse was eradicated. The eggplant isolate was able to cause severe disease symptoms on watermelon seedlings. Both isolates, the one from watermelon (36-7A) and the one from eggplant (46), were able to cause disease on tomato, eggplant, cucumber and watermelon seedlings, but not on melon, green pepper or Brassicae. Detection and identification methods for A. avenae in use and under development were discussed.

The different subspecies of A. avenae are known as pathogens of different plant species, but to the best of our knowledge this is the first report of pathogenicity on members of the family Solanaceae. (L)

Occurrence and Identification of CoUetotrichum acutatum Responsible for Strawberry Anthracnose in Israel

S. Freeman Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel

[Fax: +972-3-9683543]

The first occurrence in Israel of strawberry anthracnose caused by the fungal pathogen Colletotrichum was detected in nurseries and in the field in 1994. Morphological and cultural identification revealed the species responsible for anthracnose as C. acutatum. In 1995 most of the strawberry nurseries were heavily infected with the pathogen, which resulted in major collapse of transplants in the field. The disease was subsequently diagnosed on flowers, and green and ripe fruit in November 1995. A semiselective medium amended with iprodione at a concentration of 5 ppm was used for isolating C. acutatum from various infected plant tissues. Various molecular approaches were used for accurate differentiation among Colletotrichum species (C. gloeosporioides, C. fragariae and C. acutatum) responsible for strawberry anthracnose. Arbitrarily primed PCR (apPCR) using repetitive motif primers was the most reliable technique for rapid identification of the strawberry anthracnose pathogens. In view of the extent of crop damage, the following procedures should be considered for limiting spread of the pathogen: (i) use certified noninfected plant material; (ii) ensure sanitation measures and removal of infected plant material from the field; (iii) distance nurseries from production fields; (iv) fumigate soil and plant debris and remove weeds that may latently harbor the pathogen; (v) use regular chemical treatments and develop alternative control measures; and (vi) begin breeding plant lines for anthracnose resistance. (L)

F u n g a l Pathogens Involved in the Blossom Blight Phenomenon in Avocado cv. ' H a a s '

Miriam Zilberstaine, x Edna Levy, 2 I.S. Ben-Zeev 2 and Yael Dlugatch 3 1Ministry of Agriculture, Extension Service, Hadera 38364 [Fax: +972-6-341033];

2Ministry of Agriculture, Plant Protection and Inspection Services, Bet Dagan 50250; and aTechnological College, Petah Tiqwa 49453, Israel

The phenomenon of blossom blight of avocado cv. 'Haas' has been known in Israel for the last 15 years. It was first observed in Western Galilee in the noah, later in the center of the country, and


in the last 2-3 years in the less rainy western Negev in the south. The symptoms of the phenomenon are: blossom blight or dropping of young fruits, followed by dieback of the affected branches. Trees affected for several seasons develop smaller leaves and shorter internodes than unaffected trees. The name given to this disease, the phenomenon, reflects the lack of knowledge about its causes. Several biotic agents were suspected, mainly the fungi Alternaria alternata, Cladosporium spp., Lasiodiptodia theobromae and Hendersonula torutoidea, which were the ones most frequently isolated from symptomatic branches. Suspicions that the causal agents were abiotic factors such as polluted water, slower root activity during blossom and/or deficiencies or excesses of micro- or macroelements, were even stronger.

Colletotrichum gloeosporioides was isolated in 1993 from blighted blossom and branch tissue of cv. 'Haas' avocado with incipient dieback. This fungus is not easily isolated from these tissues, as the aforementioned fungi are quick to invade and colonize them. A literature search showed that C. gloeosporioides is the causal agent of blossom blight of citrus in Belize (a relatively new disease described in the late 1970s) and of mango in Malaysia. These facts and the common knowledge that this fungus is widespread in avocado orchards in Israel and is responsible for avocado fruit-rot in storage, led us to investigate the possible role of C. gloeosporioides as the cause of the phenomenon. A preliminary experiment, involving artificial inoculation of young, blossoming avocado trees, by spraying with a conidial suspension of C. gloeosporioides, was carried out under controlled glasshouse conditions in 1994. The symptoms obtained were similar to the phenomenon observed in the orchard and the fungus was reisolated from the infected branches. This apparently satisfies Koch's postulates, but additional inoculation experiments are needed in order to prove that C. gloeosporioides is indeed the biotic agent of this disease.

During 1994/95 a number of C. gloeosporioides isolates were obtained from blossom branches of Haas avocado with blight or dieback. These isolates are being tested now for their ability to induce blossom blight. They are also being characterized by their fatty acids profiles, determined by extraction of the fatty acids from submerged shake-cultures and analysis of the extracts by gas chromatography. The fatty acids profiles obtained indicate significant variability among the isolates, as found also by DNA profiles (S. Freeman, pers. commun.). We have still to improve the shake-culture conditions in order to ascertain that the observed variability is not an artifact of in vitro culture. More artificial inoculation experiments will be performed, under various regimes of temperature, relative humidity and simulated rain, in order to clarify the possible roles of these conditions in the onset of infection. (L)

B: BIOLOGY OF FUNGAL DISEASES

Almond Anthracnose in Israel and California

E. Shabi, 1 S. Freeman, 1 Talma Katan 1 and Beth Teviotdale 2 1Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel

[Fax: +972-3-9683543]; and ~ Kearney Agricultural Center, Parlier, CA 93648, USA

Fungi of the genus Colletotrichum cause anthracnose disease in many crops. In Israel, the fungal species C. gtoeosporioides infects various crops such as almond, avocado and (in the past) apples. Almond anthracnose was first discovered in Israel approximately 20 years ago and caused serious damage to orchards in the north and the south of the country. The fungus infects young fruit and causes wilting of leaves and dieback of branches. Almond anthracnose is distributed in the Mediterranean region and South Africa and was first reported in California in 1916. The disease was re-discovered in California in 1992 and during the spring of 1995 many almond orchards were infected by anthracnose. Isolates of the almond anthracnose pathogen from California were compared with those from Israel. Comparisons were done by measuring growth of cultures on medium amended with benomyl at concentrations of 0.05, 0.5 and 5.0 #g/ml and by molecular

138 Meetings

methods. It was found that the sensitivity of Californian isolates to benomyl did not differ from that of the Israeli isolates. However, the two populations differed in colony color, optimal growth temperatures and conidial morphology. DNA extracted from the Californian isolates showed a different profile compared with that of the Israeli isolates after PCR amplification. These findings suggest that the Californian isolates are different from the Israeli isolates, and are more closely related to the species C. acutatum than to C. gloeosporioides. Preliminary results showed that 12 isolates from California belong to a single vegetative-compatibility group. (L)

Vegetative-Compatibility Groups in Populations of Fusarium oxysporum in Israel

Talma Katan Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel

[Fax: +972-3-9683543]

Vegetative compatibility is used to classify isolates of Fusarium oxysporum according to their ability to form inter-strain hyphal anastomoses and stable heterokaryons. Isolates that form heterokaryons with one another are considered genetically related and assigned to the same vegetative-compatibility group (VCG). Strains within a given VCG have been found to be usually of the sameforma specialis (f.sp.), whereas afsp. may contain one or more VCGs. Many VCGS of E oxysporum have been given serial numbers (four or five digits) in an internationally accepted numbering system, whereas other VCGs have not yet been numbered. Following is a summary of studies of someff.sp, of E oxysporum occurring in Israel.

Eo. lycopersici, causing Fusarium wilt of tomato: a single VCG (0030) includes races 1 and 2; it occurs worldwide. Eo. radicis-lycopersici, causing Fusarium crown-and-root rot of tomato: five VCGs, three major (VCG 0090, 0091, 0092) and two minor (VCG 0093, 0096); VCGs 0090 and 0091 occur worldwide. Eo. vasinfectum, causing Fusarium wilt of cotton: a single VCG (0113) specific to race 3 in Israel, Egypt and Sudan. Eo. dianthi, causing Fusarium wilt of carnation: the major VCG 0021 is specific to race 2 in Israel and elsewhere worldwide; one isolate of VCG 0020, known to be specific to race 4 from every source tested so far. Eo. melonis, causing Fusarium wilt of muskmelon: two VCGs: VCG 135 is comprised of race 0 (occurs throughout Israel, and is reported also from France) and race 2 (Bet Netofa region, Israel); VCG 0138 is composed of race 1 (occurs nationwide) and race 1-2 (Bet Netofa region). Eo. basilici, causing Fusarium wilt and crown rot of sweet basil: a single VCG (suggested number: VCG 0160) includes all isolates tested from Israel, Italy and the USA. In preliminary studies, single VCGs were identified in additional ff.sp.: F.o. niveum, causing Fusarium wilt of watermelon (presumably VCG 0082 specific to race 2); Eo. melongenae, causing Fusarium wilt of eggplant (suggested number: VCG 0170); and Eo. cucumerinum, causing Fusarium wilt of cucumber (suggested number: VCG 0180). (L)

Fusarium oxysporum of Jojoba

Leah Tsror (Lahkim), 1 Marina Hazanovsky, 10 r l y Erlich I and I. Kosto 2 1Dept. of Plant Pathology, ARO, Gilat Regional Experiment Station, M.P. Negev 2, 85410

[Fax: +972-7-926337]; and 2Extension Service, Ministry of Agricuhure, Negev Region, Be'er Sheva 84100, Israel

Wilt and death of jojoba plants in new plantations which originated from vegetative propagation, are usually detected at the end of the second or third year after planting. The disease occurs suddenly and develops rapidly in the plantation, sometimes affecting 50-70% of the trees within a few months. The causal agent of the disease is Fusarium oxysporum, which can be isolated from the vascular bundles of the plant. The pathogenicity of several E oxysporum isolates was examined by inoculation of cuttings and seedlings. Wilt symptoms were detected in inoculated cuttings within 3 weeks. Wilt

Phytoparasiuca 24:2,1996 139

was expressed first in tbp leaves, and eventually all leaves dried up and abscised. Wilt symptoms were found on seedlings 2 years after inoculation, and included chlorosis of leaves, defoliation and plant death. F. oxysporum was re-isolated from both inoculated cuttings and seedlings. Disease symptoms of inoculated seedlings (grown from seeds) set in 6-18 months after inoculation. Jojoba seedlings (grown from seeds) and cuttings were inoculated with other Fusarium isolates which originated from different crops: E oxysporum f.sp. vasinfectum from cotton, E oxysporum f.sp. dianthi from carnation and E oxysporum f.sp. basilicum from sweet basil. None of these seedlings or cuttings showed symptoms. Cotton and tomato seedlings were inoculated with several virulent F. oxysporum isolates from jojoba; none of them showed wilt or defoliation symptoms. These findings indicate a high degree of specificity of the Fusarium isolates from jojoba.

Occurrence of the disease at different locations in plots where jojoba had never been grown indicates the possibility of latent infection in the vegetative seedlings prior to their planting in the field. In old plantations grown from seed, no Fusarium infections were observed; in the new plantations where only male plants are grown from seed, just one case was detected. Fusarium in new jojoba plantations (1992/93) was detected at different intensities. Several jojoba varieties, such as 64, XX and 100, seem to be highly sensitive to this disease. One plantation with a high incidence of the disease was abandoned. In other locations, dead plants are removed with roots intact to prevent dissemination of the fungus. (P)

Development of Gray Mold in Sweet Basil

Galit Sharabani, 1'2 D. Shtienberg, a Y. Elad, 1 A. Dinoor 2 and H. Yunis 3 1Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250 [Fax: +972-3-9683543];

ZDept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100; and 3Extension Service, Ministry of Agriculture, Tel Aviv 61070, Israel

The cultivated area of herb plants has increased markedly in recent years in Israel. The leading crop is sweet basil (Ocimum basilicum), most of which is exported, mainly to the European community. Sweet basil is severely infected by gray mold, caused by Botrytis cinerea. The pathogen may develop on the packed bunches during shipment to the target market, resulting in rot of the entire package. However, more severe damage is caused when the pathogen develops in the greenhouse, and during severe epidemics the entire crop may be devastated. The demand for products free of pesticide residues forces the growers to avoid unnecessary application of fungicides for the suppression of gray mold. Many growers do not apply any fungicides and tolerate the losses imposed by the pathogen. The objective of this research was to study the epidemiology of gray mold in order to develop an efficient agrotechnical procedure for its management.

Observations of disease development in commercial greenhouses revealed that infections occur on the stem cuttings, soon after harvest. The disease then progresses on the stem, killing all leaves and secondary buds. When it reaches the main stem, at the bottom of the plant, the entire plant dies. It was noticed that disease outbreaks occurred:w.hen harvesting was carried out during rainy periods, but not when harvesting was done a few days before or after rains. The reason for this was studied in a set of experiments conducted under controlled environment conditions, in growth chambers and in greenhouses. First, changes in host receptivity to infection, as a function of the time from harvest,. were studied. The stem cuttings were highly receptive to infection soon after harvest, but receptivity diminished gradually with time, and by 48 h after harvest most of the stem cuttings we tried to infect with spores of B. cinerea, remained uninfected. Observations under a scanning electron microscope revealed that an opaque layer developed over the cut area within a few hours after harvest. Five days after harvest, the cut area was evenly covered with that layer. Conidia of B. cinerea germinated and penetrated directly to the cut area of the stem soon after harvest, whereas they did not germinate when applied 5 days after harvest.

140 Meetings

Assuming that optimal conditions for B. cinerea infection prevail in the greenhouse when harvesting is done during rainy periods, and based on the above findings, we hypothesized that refraining from harvesting during rainy days might reduce markedly the incidence of gray mold. This hypothesis was tested in an experiment conducted in an experimental greenhouse. It was found that harvesting 5 days after heavy rains reduced significantly (by 75%) the incidence of gray mold in comparison with plots harvested one day after or during the rains. The conclusion is that it may be possible to reduce the losses induced by gray mold in basil by appropriate timing of the harvests. (P)

Genetic Diversity in Leaf and Stripe Rusts in Israel

J. Manisterski, 1 Pnina Ben-Yehuda, 1 E. Kosman a and Z. Eyal 1,2 l lnst. for Cereal Crops Improvement, and 2Dept. of Botany, The George S. Wise Faculty of Life Sciences [Fax: +972-3-6409380] and 3 School of Mathematics, The Faculty of Exact Sciences,

Tel-Aviv University, Tel Aviv 69978, Israel

Isolates of wheat leaf rust (Puccinia recondita f.sp. tritici) and of wheat stripe rust Puccinia striiformis) originating from wheat fields in Israel during the years 1993-1995, were tested on seedlings of wheat differentials. The genetic diversity was estimated within and between years. The frequency of virulence among leaf rust isolates increased on the differentials possessing the genes Lrl, Lr2a, Lr3 and Lr 26, and decreased on Lrl 1, Lrl5, Lrl7, Lr21 and Lr30. Genes Lr9 and Lr24 were resistant to the 160 tested isolates, whereas Lrl0, Lrl8 and Lr23 were susceptible to all the isolates. The highest genetic diversity among isolates was recorded in 1994; diversity increased significantly from 1993 to 1994. Genes Lrl, Lr2c, Lr3, Lrl5 and Lrl7 differentiated between the isolate populations in the different years.

The frequency of virulence of stripe rust isolates on the gene Yr9 continued to be high on wheat differentials possessing the 1BL/1RS translocation from rye. Virulence on 'Dariel' in 1995 was as high as in 1994, when it was observed for the first time in Israel. Virulence on Dariel was confined in 1994 to northern Israel, whereas in 1995 it was widely distributed in wheat fields across Israel. The virulence spectra of other isolates collected prior to 1994, and during 1994 and 1995, showed a high level of stability. (L)

The Use of Flow-Cytometry for Fungal Identification

Tamar Eilam and Y. Anikster l l n sL for Cereal Crops Improvement and 2Dept. of Botany, The George S. Wise Faculty of Life

Sciences, Tel-Aviv University, Tel Aviv 69978, Israel [Fax: +972-3-6409380]

Quite frequently it is necessary to distinguish between very close fungal types, subspecies and species. When the morphological resemblance of tested spores is very high or differences in host- range of these fungi are minor, it is difficult or sometimes impossible to differentiate according to morphological parameters and host range. The use of a flow cytometry sorter (FCS) may solve the problem, the system-tested parameters being relative nuclear DNA content and nuclear size. The FCS is. suitable for measurements of symmetric spores that are separated from hyphae.

In our research on the various types of wheat leaf rust - Puccinia recondita, we estimated the relative content of the nuclear DNA, compared with P. hordei as standard. We tested pycniospores, as these spores are the only ones in the rust life cycle which are both single-celled and mononucleated. Each pycnial cluster contains many tens of thousands of genetically identical spores which have originated from one basidiospore. Spores were stained with propidium iodide (PI), a fluorochrome which intercalates quantitatively to DNA. 10,240 pycniospores were tested in each run. The relative fluorescence intensity of PI-stained pycniospores nuclei of rust species was: for Puccinia graminis,


56 (the genome of P. gramin& contains 67 - 106 base pairs); P. recondita tritici, 75; P. hordei, 100; P. recondita variabilis type, 130; Tranzschelia pruni-spinosae, 150; P. helianthi, 185; and Uromyces appendiculatus, 346. It is clear that the two P. recondita types have different amounts of nuclear DNA. These results are a negative answer to the question "Are the Aegilops leaf rusts the primary foci for the annual attacks on cultivated wheats in Israel or other countries in our region?" (L)

C: SOILBORNE FUNGAL PATHOGENS AND THEIR CONTROL

Phytopathological and Physiological Aspects of Sudden Wilt of Melons in the Arava Region

S. Pivonia, 1 R, Cohen, 2 I.S. Ben-Ze 'ev , 3 U. Kafkafi 4 and J. ,Katan 5 1Arava Research and Development, Sapir Center 86825; 2Dept. of Vegetable Crops, ARO, Newe

Ya'ar Research Center, Haifa 31900 [Fax: +972-4-9846936]; 3Ministry of Agriculture, Plant Protection and Inspection Services, Bet Dagan 50250; 4Dept. of Field and Vegetable Crops and Genetics and 5Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem,

Faculty of Agriculture, Rehovot 76100, Israel

Sudden wilt of melons is a destructive disease which is a major problem in the Arava region of southern Israel. The disease is also known and being investigated in Spain and in the southern the United States. The disease causes rapid wilt of the plants and may lead to total loss of the yield. Some soilborne fungi such as Fusarium, Pythium, Rhizoctonia and Monosporascus were isolated from wilted plant roots sampled from methyl bromide-treated fields in the northern and central Arava. Monosporascus was the main fungus isolated from non-fumigated fields. It has been reported to be one of the causal agents of sudden wilt in melons in various parts of the world.

In an inoculation experiment conducted under field conditions in the Arava, 72% of plants inoculated with Monosporascus and 23% of those inoculated with Pythium, wilted 70 days after planting; inoculation with a combination of Pythium and E solani resulted in increased wilt incidence (58%) compared with a low wilt incidence with each pathogen alone. Disease severity and the time at which symptoms were observed were similar in the Monosporascus-inoculated plants and in plants which developed in naturally infested soil from En Tamar and En Yahav. The sudden wilt syndrome was associated with environmental stresses and with stress induced by maturation of the fruits. To confirm the latter correlation, a field trial in which fruits were removed was conducted. Normal plants with an average of 2.5 fruits per plant reached 98% mortality. Plants that were thinned to one fruit, or plants with no fruits, had an incidence of 75% and 12% wilt, respectively. The relationship between dry weight accumulation and time of wilt symptoms appearance was tested in a methyl bromide-treated soil and in an infested soil. The time at which foliage growth stops and fruit weight is increasing was correlated with the beginning of the wilting. (L)

Grafting: A Possible Solution for the Sudden Wilt Disease in the Arava Region

M. Edelstein, 1 R. Cohen a and Rivka Offenbach 2 1Dept. of Vegetable Crops, ARO, Newe Ya'ar Research Center, Haifa 31900

[Fax: +972-4-9846936]; and ZArava Research and Development, Sapir Center 86825, Israel

Sudden wilt of melons (Cucumis melo L.) is a destructive disease and a major problem in the Arava region of southern Israel. Soil disinfestation by methyl bromide fumigation is the common' treatment used in that region to overcome the problem. The threat of limitation in the use of this chemical motivated researchers to seek alternatives. Grafting is a technique widely used in some countries in the Mediterranean Basin. In Israel, this method is not used because of the ready availability of methyl bromide. Six rootstocks of Cucurbita were evaluated at two locations in the

142 Meetings

Arava. In one experiment conducted at En Tamar, four rootstocks gave satisfactory results. Grafted plants reached the end of the growing season with no wilt symptoms and with good quality fruits. Two other grafted combinations suffered approximately 50% mortality, and 95% of the non-grafted cv. 'Arava' plants were destroyed by the disease. In another experiment conducted at En Yahav, the same combination of plants was planted. Rootstocks that excelled at En Tamar had approximately 50% mortality at En Yahav and reached the end of the season without fruit and in very poor condition. It seems that differences in the inoculum level in the soil or other factors may have contributed to the differences between the two locations.

Horticultural parameters were compared between grafted melon and watermelons and non- grafted plants in soil free of pathogen at Newe Ya'ar (in northern Israel). No differences in yield, fruit size or total soluble solids were found between grafted and regular plants.

In the future, the research will concentrate on searching for rootstocks with resistance to a wide range of pathogens and on adapting new agrotechniques for grafted plants. (L)

Fusarium Wilt and Crown Rot of Sweet Basil: Identification, Epidemiology and Control

A. Gamliel, 1 Talma Katan, 2 H. Yunis 2 and J. Katan 3 1 Laboratory for Research on Pest Management Application [Fax: +972-3-9604704] and 2Dept. of

Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250; a Extension Service, Ministry of Agriculture, Tel Aviv 61070; and 4Dept. of Plant Pathology and Microbiology, The Hebrew

University of Jerusalem, Faculty of Agriculture, Rehovot 76100, Israel

Fusarium oxysporum f.sp. basilici (Fob) causes wilt and crown-and-root rot in sweet basil in all basil-growing regions of Israel. Wilting and death of basil plants were recorded, and isolates of E oxysporum were obtained at high frequency from roots and all above-ground parts of diseased plants. Inoculation tests of representative isolates verified that the causal agent is Fob. This species causes both wilt and crown-and-root rot symptoms, of which we observed the following: wilt of plants at all stages of growth, brown and black discoloration of roots and lower stems, continuous and discontinuous black lesions on the stems, blackening and drying of vegetative apices, growth retardation and xylem discoloration. The stems were frequently covered with a pink-orange layer consisting mainly of macroconidia which could become airborne, as revealed by propagule trapping. Therefore, the pathogen has characteristics of both soilborne and airborne pathogens. Conidia of the pathogens were trapped in a basil greenhouse during the growing seasons, in amounts significantly correlated with disease incidence of the plants. Fob was readily recovered from structures inside the greenhouse including concrete poles, strings, pipes, and walls. Seeds collected from diseased plants were infected with the pathogen and produced diseased plants bearing infected seeds.

Soil disinfestation alone was partially effective in controlling the disease, the progress of which was suppressed during only the first growth stages. Soil disinfestation combined with solarization of the greenhouse structure, including all the irrigation equipment, gave effective control of the disease throughout the season. Since the pathogen is easily disseminated, a holistic approach for its control is necessary. Effective control of the disease can be achieved by combined soil and structure disinfestation together with using pathogen-free seeds and transplants. (L)

Airborne Inoculum of Fusarium oxysporum f.sp, lycopersici in Greenhouses of Cherry Tomatoes

Talma Katan, 1 E. Shlevin 2 and J. Katan 2 1Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250 [Fax: +972-3-9683543];

and 2Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of Agricuhure, Rehovot 76100, Israel


Plants exhibiting symptoms of wilt and xylem discoloration, which are typical of Fusarium wilt caused by Fusarium oxysporum f.sp. lycopersici (Fol), were observed in greenhouses of cherry tomatoes (cv. 139). Some plants, which were at advanced stages of disease development and desiccation, had their lower stems covered with a thin, light pink layer of macroconidia of F. oxysporum. This sign resembles the sporulating layer on stems of tomato plants infected by E oxysporum f.sp. radicis-lycopersici (Forl), which causes the crown and root rot disease in tomatoes, and was observed in many greenhouses throughout the country. Monoconidial isolates were used in pathogenicity and vegetative compatibility tests. The isolates were pathogenic on tomato cv. 'Marmande' but not on cv. 'Rehovot-lY, thus identifying the pathogen as Fol race 1. The isolates were assigned to vegetative-compatibility group 0030 of Fol. The possibility of coinfection with Fol and Forl was excluded by testing several macroconidia from each plant. Airborne propagules of Fol were trapped on Fusarium-selective medium in greenhouses in which plants with a sporulating layer were growing. The phenomenon was reproduced by inoculating tomato plants (cvs. 139 and Marmande, both susceptible to race 1 of the pathogen) with race 1 of Fol, whereas only certain isolates of race 2 of Fol produced a sporulating layer on artificially inoculated plants. This phenomenon has not been reported previously with Fol and might be connected with specific environmental conditions, e.g. high humidity in the greenhouse, or with certain cultivars. The sporulation of Fol on plant stems and dissemination of macroconidia by air movement may have serious epidemiological consequences. Sanitation of the greenhouse structure, e.g. by space solarization, along with soil disinfestation and other means, is necessary in order to ensure effective disease control. (L)

Organic Soil Amendments as an Alternative Way to Control Soilborne Pathogens

G. Kritzman 1 and G. Lazarovits 2 1Dept. of Plant Pathology, ARO, The Volcani Center~ Bet Dagan 50250, Israel

[Fax: +972-3-9683543]; and 2Agriculture and Agri-Food Canada, Pest Management Research Centre, Ont., Canada N6G 2VE

Experiments were carried out during 1994 and 1995 in Canada in the laboratory, in the greenhouse, in micro-plots on Sandford farm, and in potato fields (Simcoe and Alliston area). Soils of fields heavily infected with Streptomyces spp., the causal agents of potato scab, were chosen. The soils were mixed with one of the following organic amendments on a weight-to- weight basis: blood meal, feather meal (1%), or soy meal (2%). All three materials tested resulted in similar major changes in the population of soil microorganisms, increase of certain groups and suppression of others. Microsclerotia of Verticillium dahliae that were buried in the soil and recovered at various times after burial, were completely eradicated within 14 days. The Streptomyces populations were similarly reduced to a barely detectable level within the same time. The large weed population that sprouted after about one week was found to be destroyed within 14 days and there are good indications that the population of nematodes was also affected. No such changes were seen in untreated control soils. Potato tubers were planted about one month after soil amendment incorporation. Tubers that were harvested from plants in amended soils were completely free of any scab symptoms, whereas in the untreated plots, only approximately 10% of the tubers were healthy. Similar results were obtained in the same plots after a second crop. It seems that two major processes took place in the treated soils. The first step is ammonification of the proteins in the amendments and the second step is creation of a suppressive soil by changing the original soil population and increasing the total microorganisms in the soil; this phenomenon is still under investigation. (L)

144 Meetings

Suppression of Soilborne Pathogens by Composted Municipal Solid Waste

R. Cohen, i N. Bar-Lev, 1 B. Chefetz, 2 Y. Chen 2 and Y. Hadar i 1Dept. of Plant Pathology and Microbiology [Fax: +972-8-9466794] and 2Dept. of Soils and Water,

The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100, Israel

Compost is the stable product of aerobic microbial decomposition of heterogenic organic matter. Various organic wastes such as grape marc, cattle manure, hardwood bark, yard waste, municipal solid waste (MSW) and sewage sludge can be used as raw materials for composting. Composting of the organic fraction of MSW can reduce up to 50% of the waste volume arriving at the landfills and produce a stable product that can be used in agriculture. Organic matter decomposition level and maturity of compost affect the ability to suppress soilborne pathogens. In recent years, compost amendments in agriculture have been investigated for use in an integrated system of biological control due to their ability to suppress soilborne plant pathogens including Pythium aphanidermatum and Rhizoctonia solani. The objectives of this work were to study the suppressiveness of composted- MSW (CMSW) against soilborne plant pathogens and to achieve an understanding of the microbial mechanism involved in the suppression.

The suppressiveness of CMSW (20% organic matter) to P aphanidermatum, which causes damping-off in cucumber, was studied in the greenhouse in polypropylene pots (500 ml) containing compost, peat and vermiculite (1:1:1, v/v/v). CMSW reduced disease incidence on plants by approximately 50%, and in sand (containing 20% CMSW) by 70% vs control. In addition, in experiments in which the suppressiveness against R. solani in cotton was studied in sandy soil amended with 5-20% CMSW, the disease incidence was reduced by approximately 40% vs control. Autoclaving (two times, 1 h each, 121~ gamma irradiation (5 Mrad) or heat treatment (5 days at 60~ of CMSW, eliminated the suppression ability. Thus, it may be concluded that the mechanism involved in suppression of R. solani, is biological. Another proof of involvement of microorganisms was obtained by addition of 5% (v/v) CMSW to heat-treated CMSW. This addition caused the recolonization of antagonist microorganisms and the return of suppression ability to the compost. In the case of R. solani the mechanism involved in the suppression is presumed to be lysis of fungal hyphae. Microscopic examination of R. solani incubated in CMSW-amended soil indicated the possible involvement of bacteria. Through understanding the microbial mechanism involved in suppression of soilborne pathogens, the quality of compost can be improved by enriching the latter with beneficial microorganisms The key to large-scale utilization of disease-suppressive compost is the development of reproducible products with defined and consistent properties. (L)

Suppressive Soils against Root Pathogens in Israel

Lea Madi and J. Katan Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem,

Faculty of Agriculture, Rehovot 76100, Israel [Fax: +972-8-9466794]

Suppressive soils are those which suppress disease development or reduce pathogen population, in contrast to conducive soils. Assays to determine level of suppressiveness of soils before planting as well as for using these soils as a source for isolation of effective antagonists, were conducted. We screened natural soils from different areas of Israel (Ze'elim, Re'im, Nir Oz, Rehovot) and soils treated with methyl bromide or solarization, for their capacity to suppress Pythium aphanidermatum, Sclerotium rolfsii and Rhizoctonia solani in greenhouse experiments. We examined different parameters of pathogen growth, including germination, rate of linear growth and hyphal density, in the tested soils. Our assays revealed a negative correlation (r=0.876) between germination strength


of S. rolfsii sclerotia and suppressiveness of the soil to S. rolfsii. No significant correlation was found with the other parameters tested. Disease reduction in some of the suppressive soils was ,~, 50% when tested with P. aphanidermatum and S. rolfsii and up to 80% with R. solani. "),-irradiation and dilutions of the suppressive soils decreased the suppression level, indicating the involvement of microbial activity in suppression. Microbial analyses of the suppressive soils revealed large populations of Pseudomonas and Penicillium in some of the tested soils. We isolated several potential antagonists from the suppressive soils. Addition of one of these microorganisms to a conducive soil increased the suppression level of that soil to 50% against S. rolfsii and to 40% against R. solani. These studies indicate that soil suppressiveness is connected, to some extent, with biotic factors. (P)

Pathogen Killing Rates under Fluctuating Temperature and Moisture Regimes during Space Solarization

E. Shlevin, 1 J. Katan, 1 Y. Mahrer 2 and P. Di Primo 1,3 1Dept. of Plant Pathology and Microbiology [Fax: +972-8-9466794] and 2Dept. of Soil and Water Sciences, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100, Israel; and

a lnst. of Plant Protection, University of Reggio Calabria, 89061 Gallina, Italy

Soil disinfestation, even if very effective, does not prevent recontamination of the soil by inoculum surviving on the greenhouse structure. Space solarization, namely, disinfesting the greenhouse space and structure, by closing and by heating it by solar irradiation, is a complementary control action which aims to prevent contamination of the soil or the plants by the surviving inoculum. Space solarization is carded out by closing the greenhouse during summer time, thereby raising air temperatures to 55-65~ This is a nonchemical, simple and inexpensive method for sanitation that involves dry heating, which is, however, less effective in pathogen control than wet heating. The aims of the present study were to quantify the rate of pathogen killing under space solarization, to develop models for predicting it according to the climatic data of the closed greenhouse, and to improve pathogen control. The pathogens studied were Sclerotium rolfsii (sclerotia) and Fusarium oxysporum f.sp. radicis-lycopersici (macroconidia and chlamydospores).

Rate of pathogen mortality was expressed as a proportion of mortality at various periods of exposure (in days), or accumulated hour-degrees for temperatures above 45 ~ 50 ~ or 55~ (accumulated number of hours above each of the indicated temperatures). A logistic model was adapted. When the independent variables were hour-degrees above 45 ~ and 50~ a high coefficient of determination (R z) of >0.85 was obtained in most cases.

Wetting the greenhouse by means of sprinkler irrigation did not improve pathogen control, due to cooling of the inoculum by evaporation. Results from simulation experiments suggest that cooling was caused by contact of the hot and humid air inside the greenhouse with the cooler plastic cover, resulting in condensation of water vapor, continued water vapor deficit, enhanced evaporation and finally in cooling of greenhouse space.

Repeated experiments demonstrated that space solarization for greenhouse sanitation is an effective tool for controlling a variety of pathogens. (L)

D: SOIL TREATMENTS: METHYL BROMIDE AND ITS ALTERNATIVES

Methyl Bromide: Current Status and Future Trends

J. Katan Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of

Agriculture, Rehovot 76100, Israel [Fax: +972-8-9466794]

The fumigant methyl bromide (MB) is the major soil disinfestant in Israel as well as the whole world over, especially in intensive agriculture. This is due to its broad spectrum of activity, as well

146 Meetings

as to other advantages. MB was recently declared an ozone-depleting substance, although there is controversy regarding its impact. This development and the general tendency to reduce the use of all pesticides, led to efforts to reduce MB consumption and finally to phase it out. According to the recent Vienna meeting, its consumption has to be reduced according to a timetable. A phase- out is scheduled for the year 2010, with the exception of certain critical uses and consumption in developing countries. In Israel, teams appointed by the Ministers of Environment and of Agriculture are dealing with many aspects of MB. The general strategy is to reduce MB use without diminishing pest control effectiveness. This can be achieved through two parallel and complimentary approaches: (i) In the near future, to develop improved technologies in order to reduce MB dosages without reducing pest control effectiveness, e.g. by using impermeable plastics, combining different methods of control and using improved methods of application. (ii) In the long run, to develop new nonchemical and chemical alternatives, or to expand the use of already existing ones. The accomplishment of the latter task is difficult but should be attempted. Certain intensive crops depend heavily, at this stage, on the use of MB. The too rapid and unrestrained introduction of new methods of control may lead to severe agricultural and environmental consequences. The development of the above strategy requires concentrated and coordinated efforts in research, extension, education, legislation and financial support. (L)

Reduced Methyl Bromide Emission by Improved Technology

A. Grinstein, 1 A. Gamliel t and J. Katan 2 1Laboratory for Research on Pest Management Application, ARO, The Volcani Center,

Bet Dagan 50250 [Fax: +972-3-9604704]; and 2Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100, Israel

Methyl bromide (MB) is a fumigant used mainly for soil disinfestation against a wide array of pests and diseases. It is an important disinfestant of agricultural durables and perishable products. Disinfection of structures, stored commodities and transportation vehicles, for direct use on agricultural products and in quarantine, are other important uses. The Montreal Protocol listed MB as a potential ozone depletor. Research efforts were therefore initiated to develop improved application techniques to facilitate MB use, while concomitantly reducing its emissions to the atmosphere.

Most of the MB produced (,,~ 75%) serves for soil disinfestation. The rest is consumed for various other purposes. Technologies for recovery and recycling of MB from treated enclosures were developed and can be implemented.

Application of MB for soil disinfestation is manual or mechanical. MB is injected as a liquid or spread as gas over the treated area. Tarping with plastic sheets, mainly polyethylene (PE), prevents rapid aeration. The fumigated area is continuous, or in 0.80-1.20-m-wide strips. Some mechanical 'deep' injection to the ~80-cm-soil depth without covering the area is done in the USA.

PE is permeable to MB, and its permeability increases with temperature. Permeability rates of 5 g MB /m2/h were recorded in Israel. MB emission occurs also from the edges of the sheeting, resulting in reduced efficacy (the 'border effect'). The estimated emission resulting from the accepted techniques can be as high as 50-60%; the rest of the MB remains in the soil, mainly as bromide salts. The recommended dosages are normally higher than those actually needed, both due to the use of PE and to the need for maintaining a lethal level of MB throughout the treated area, including near the borders as well as at loci that suffer from only partial MB penetration.

Reduced emission of MB is achieved by preventing its leakage from as many sources as possible. American researchers showed that increasing the distance between the injection shanks was efficient in reducing MB emissions in the deep application.

Replacing the PE sheeting with virtually impermeable films enables a reduction of 50-60% in the recommended dosages. Permeability of films based on ethylene-vinyl alcohol, for example, is only 10 -4 of that of PE films. In addition, such films effectively decrease the emissions along the borders,


when the sheet edge is incorporated deep in the soil. Continuous mulching reduces the proportion of the border to the plot, as well as the proportion of MB emission. Uniform distribution of MB in the plot can result in decreased dosages, by obviating overdoses at loci that are penetrated by MB only with difficulty.

Improved MB penetration to deeper soil layers by better cultivation, adjustment of soil humidity and a decrease of the recommended dose at higher soil temperatures allow further reduction of the emissions. Flexible recommendations according to the pests in the plot and prevention of application errors by the operator can further reduce dosages as well as emissions. (L)

For-Dor 37, a Soil Treatment Product to Control Soilborne Diseases

I. Peretz Alon, x O. Heiman, 2 Z. Bar 1 and E. Serlin 1 1Agricultural Committee, Ma'on District Settlements, M.P. Negev, 85465; and

2 Dor Chemicals Ltd., Haif a 26110, Israel [Fax: +972-4-8415194]

For-Dor 37 is a product intended for soil treatment against soilborne diseases in various crops. It is registered as a preplant soil treatment for growing potatoes, peanuts and tomatoes and is in the process of being registered for carrot. The active material is formaldehyde, which eventually breaks down in the soil to the innocuous compounds CO2 and water. The compound had served for soil sterilization already in the 1920s, mainly as a nematicide.

The main difficulty lies in efficient and safe application of the product. In the late 1980s, the feasibility of adapting the product for use against bacterial diseases, especially against Streptomycetes (Streptomyces spp.), causal agents of scabs, such as common scab; deep-pitted scab of potato; and pod wart disease of peanuts, was investigated. The preparation was effective also against tomato canker (Clavibacter michiganensis) and tomato pit necrosis (Pseudomonas corrugata). Later, the effect of the product against a series of soil-inhabiting fungi and nematodes was investigated.

At the beginning of the 1990s, a specialized structure to apply the material was built. It includes a system for spraying and soil incorporation within a drift-preventing structure. The structure enables application of the product and its soil-incorporation at the required depth in a single step, in the least time, to minimize drift. In order to improve the less-than-satisfactory uniformity, trials were conducted to apply the product by the standard technique of chemigation used for another soil- sterilizing product, metam-sodium (Vapam; Edigan), viz., through an irrigation system separate from the drinking water supply system. Early in the trial, the dosage and the application technique were adapted for the product. Greatest efficiency was achieved when the product was made to penetrate the soil during irrigation equally and uniformly to the correct depth. This brought about improvement, mainly in the quality of the crop and, in many cases, also in yield gain. These results were verified in semicommercial trials and observations. (L)

The Use of 'Trichoderma 2000' for Control of Plant Diseases

O. Kleifeld, 1 I. Chet, 2 Miriam Abramsky, 2 Iris Yedidia 2 and Agron Ltd. 3 1Mycontrol Ltd., Alon haGali117920 [Fax: +972-4-9861827]; ~ Dept. of Plant Pathology and

Microbiology, The Hebrew University of Jerusalem, Faculty of Agricuhure, Rehovot 76100; and 3Agron Ltd., Rehovot 76121, Israel

The activity of Trichoderma against plant pathogens is well known. The physiology of the fungus and its mode of action against the pathogens have been extensively investigated. It was found that different isolates of the fungus exhibit specific action against different pathogens.

Laboratory preparations of single isolates were tested in the field and gave control of pathogens which was comparable or even superior to that of the commonly used chemical fungicides. In order

148 Meetings

to obtain a product which would protect plants against several pathogens, two isolates of different specific activity were applied as a mixture in one preparation: isolate TH-35 - which is active against Fusarium spp. and Rhizoctonia solani, and isolate TH-315 - which is active against Pythium spp. and R. solani.

The mixture, 'Trichoderma 2000', was effective against four different pathogens: R. solani, Pythium spp., Sclerotium rolfsii and Fusarium spp. There was no difference between the effectiveness of the mixture and that of the single isolates when checked individually against each pathogen.

Since the method of the experimental application we had used was not appropriate for commercial scale use, application of the product to seedlings ('loading') in the nursery was investigated. Preparation 'Trichoderma 2000', which contains spores, plus nutrients for a better start of development of the fungus, was mixed into the growth medium in 'Speedling' trays. The fungus, which is inactive in the preparation, starts developing in the growth medium and on the roots of the plants in the 'Speedling' tray. After planting in the greenhouse or in the field, the fungus continues to develop in the rhizosphere and protects the plant against pathogens throughout the growth period (3-6 months). The fungus develops in the soil close to the plant roots, but a significant increase of Trichoderma was found also at a distance of 50-60 cm from the planting site.

'Trichoderma 2000' was applied in field tests to 'Speedling' trays of carnations. It brought about a significant reduction to complete prevention of R. solani and Fusarium infections throughout the entire growth period of the carnations in the commercial field. (L)

Biocontrol of Sclerotium rolfsii and Verticillium dahliae by Talaromyces flavus is Mediated by Different Mechanisms

Lea Madi, 1 Talma Katan, 2 J. Katan 1 and Y. Henis 1 1Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of

Agriculture, Rehovot 76100 [Fax: +972-8-9466794]; and 2Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel [Fax: +972-3-9683543]

Talaromyces flavus (Klocker) Stolk and Samson has been reported to antagonize Verticillium dahliae, V. albo-atrum, Sclerotinia sclerotiorum and Rhizoctonia solani. To study the mechanisms involved in biocontrol of S. rolfsii and V. dahliae, the capacity of 12 isolates of T. flavus to (i) produce chitinase,/3-1,3-glucanase, cellulase, and glucose oxidase; (ii) produce antifungal substance(s) active against V. dahliae; (iii) parasitize sclerotia of S. rolfsii; and (iv) reduce bean root rot caused by S. rolfsii, was examined. BenRTFI-R6, a mutant resistant to benomyl, was found to be a strain producing extracellular enzymes in excess that exhibits enhanced antagonistic activity against S. rolfsii and V. dahliae. Correlation analyses between the extracellular enzymes' production by T. flavus isolates and their ability to antagonize S. rolfsii, revealed a high correlation with chitinase activity. The antifungal compounds and glucose oxidase activity strongly inhibited hyphal growth, germination and melanization of microsclerotia of V. dahliae, but did not affect germination or melanization of sclerotia of S. rolfsii. Our results relate mycoparasitism and biocontrol of S. rolfsii to chitinase activity, while antifungal compounds and glucose oxidase activity may primarily play a role against V. dahliae. (L)


E: CONTROL OF DISEASES BY PHYSICAL, VARIOUS CHEMICAL, AND BIOLOGICAL CONTROL MEASURES

Field Evaluation of SDCA, a Computerized Decision Support System for the Management of Sunflower Rust

D. Shtienberg Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel

[Fax: +972-3-9683543]

Sunflower rust, caused by Puccinia helianthi, is a common disease in Israel. However, the time of disease onset, the severity of the epidemic and the resulting yield losses vary markedly among fields. When the disease appears at early stages of host development, yield losses are high, sometimes reaching 90% of the attainable yield. In a series of experiments conducted in 1991-93, a threshold for the initiation of chemical control was determined and verified. In addition, a model that predicts disease progress and a model that enables one to estimate the yield loss were developed. These findings led to the construction of a decision support system named SCDA (Sunflower Disease Control Advisory), which determines whether spray applications are needed and calculates the cost effectiveness of such measures.

The decision-making procedure followed by the system consists of five separate steps: (i) determination of the disease progress rate based on planting density; (ii) prediction of future disease progress based on the expected disease progress rate, and assuming a logistic increase in disease severity; (iii) prediction of the expected damage based on the yield loss model; (iv) analysis of cost effectiveness based on the value of the crop, the expected yield loss and the cost of spraying; and (v) recommendation of a specific action to be taken.

The SDCA system was programmed and distributed to all sunflower growers in Israel. They operated the system on their personal computers and followed the recommendations given by the system. The performance of the system was evaluated in six observations conducted in commercial fields. In these observations, part of the field was treated according to the recommendation of the SDCA system and the rest of the field was left untreated (the common practice). Disease progress and yield were determined for both parts. In general, predictions of disease progress made by the system were very close to the actual development of the disease. Moreover, in all fields where the recommendation of the SDCA system was followed, yields were increased by 150--460 kg/ha. The net increase in income (after deduction of the application costs) was US$193/ha. (P)

Hot Water Treatment as a Means to Control Postharvest Decay in Citrus Fruits: Prospects and Problems

V. Rodov, J. Peretz and S. Ben-Yehoshua Dept. of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250, Israel

[Fax: +972-3-9604428]

Heat treatments, such as curing (36-38~ 48-72 h) or hot dips, are promising non-fungicidal means to control postharvest decay of citrus fruits and to reduce their sensitivity to suboptimal storage temperatures. The morphological changes in the fruit surface caused by high temperature may be involved in the mode of action of heat in alleviating chilling injury.

The combination of a hot water dip (2 min at 51-52"C) with a TBZ (thiabendazole) drench (1000 ppm) was more effective than a SOPP (sodium orthophenylphenate) dip (0.35%, 35~ + TBZ 1000 ppm in controlling grapefruit decay during pilot packing-house trials. The combination of either curing or hot dips with one of the following: individual seal-packaging, packaging in polyethylene-lined cartons, or waxing, controlled decay of oroblanco fruit without using fungicides,

150 Meetings

i.e., similarly to the standard packing-house treatment including TBZ application (2000 ppm) and waxing. However, the risk of phytotoxic damage affects the consistency of the results obtained with hot dips and complicates the practical implementation of this technique. Hot water dip in a pilot-plant installation simulating the packing-house conveyor path, resulted in more damage than when the dip was employed in a stationary batch system. The following approaches to overcome this difficulty were discussed: optimization of treatment conditions in relation to fruit peculiarities, addition of permitted chemicals to the hot dip, and use of appropriate equipment. (L)

Reducing Carrot Decay during Storage by Using Polyethylene Perforated Linings

U. Afek, Janeta Orenstein and E. Nuriel Dept. of Postharvest Science of Fresh Produce, ARO, Gilat Experiment Station,

M.P Negev 2, 85410 Israel [Fax: +972-7-926485]

There are two main problems concerning storage of carrot (Daucus carota L.): development of dry and soft rot diseases caused by the fungi Stemphylium radicinum, AIternaria ahernata, Sclerotinia sclerotiorum and the bacterium Erwinia caratovora; and sprouting. Today, a solution of thiabendazole (TBZ) and sodium hypochlorite (chlorine) is used as a postharvest application to control these pathogens. However, use of chemicals such as TBZ is limited in many countries in Europe, and new methods are required to reduce decay in stored carrots.

Results of our study indicate that decay and sprouting of stored carrots decreased significantly when the carrots had been packed in cartons lined with micro-perforated polyethylene (1-mm-diam holes spaced 1 cm apart). Currently, carrots are packed for storage in cartons lined with polyethylene that has perforations consisting of 6-mm-diam holes 15 cm apart. After one month of storage at 0.5~ plus 7 days of shelf life at 20~ carrots packed with the micro-perforated linings showed 6% level of decay, as compared with 28% decay in carrots packed with conventionally perforated linings.

The micro-perforated linings also help to prevent sprouting. The sprouting level of carrots stored under the same conditions as described above, packed in micro-perforated linings, was approximately one-third that of carrots stored in conventional linings. (L)

Mechanical Aerosol Generator (Cold Fogger) for the Control of Powdery Mildew in Solidaster

M. Austerweil, 1 Tsilah Ben-David, 2 B. Steiner, 1 Yehudit Riven, 1 V. Zilberg, 1 R. Amir 2 and A. Grinstein 2

a Laboratory for Research on Pest Management Application, ARO, The Volcani Center, Bet Dagan 50250 [Fax: +972-3-9604704]; and 2Extension Service, Ministry of Agriculture,

Tel Aviv 61070, Israel

Techniques for automatic air-assisted pesticide application are of growing interest for expanding cultivation in plastic greenhouses. One of these techniques is 'cold fogging' - a very-low-volume application by aerosol generators (droplets <25 #m diameter), released in an air stream. The dispersion and deposition are assisted by the air movement inside the closed greenhouse. Good penetration into the foliage, uniform spray deposits and an exceptionally high ratio of upper-to-lower leaf surface deposits were recorded by cold fogging in various crops in Israel. The high efficiency of the technique has been proved in the control of different pests: Western flower thrips - Frankliniella occidentalis, adults of the sweet potato whitefly - Bemisia tabaci, and the carmine spider mite - Tetranychus cinnabarinus.

The efficacy of cold fogging application of the systemic fungicide Systhane (myclobutanil) for the control of powdery mildew in the ornamental crop solidaster was studied. Three consecutive foggings, at intervals of 4 and 11 days from the first application, controlled the disease completely,


as visually estimated. After the first fogging, the control of powdery mildew was 53-78%, but only in locations where high surface deposits were measured. After the second treatment the disease was partially controlled (50%) in the whole greenhouse (0.2 ha), whereas after the third application the disease was completely controlled. Surface deposits were determined by adding a fluorescent tracer to the spraying solution. The chemical analysis confirmed the efficient distribution of the spraying liquid and showed a good correlation between control of the disease and deposits. Due to the large diversity of diseases and plants, further studies should be conducted to establish recommendations for optimal application of the technique against other diseases and pests in different crops. (L)

Control of Black Scuff in Potatoes

Leah Tsror (Lahkim), 1 L. Livescu, 1 Marina Hazanovski, 1 0 r l y Erlich, 1 M. Aharon, 1 R. Barak, 1 I. Peretz, 2 B. Bing 3 and A.Yaniv 3

1Dept. of Plant Pathology, ARO, Gilat Regional Experiment Station, M.P Negev 2, 85410 [Fax: +972-7-926337]; 2Maon Area Settlements, Eshkol Regional Council, M.P Negev, 85350," and

a Lidorr Chemicals Ltd., Ramat haSharon 47100, Israel

Black scurf of potatoes, caused by the fungus Rhizoctonia solani K~ihn, causes cankers on the stems, necrosis in the stolons, and the formation of sclerotia on the daughter tubers, sometimes accompanied by cracks or distortion of the tubers, Injury of the stolon is the prime cause of a decrease in yield, although severe injury of the stems also affects the crop. In this study Monceren- Combi (pencycuron) was tested for control of black scurf in potatoes: as a soil treatment and as a seed-tuber treatment. Some of the Monceren-Combi (20% pencycuron + 50% captan, W.P.) treatments improved both yield level and quality: application of 5 kg/ha in the planting bed increased the yield significantly. Dusting and application in the planting bed with a lower concentration of Monceren-Combi (2.5 kg/ha) were also effective and brought about an increase in yield. By contrast, a dip treatment (2% Monceren-Combi) of seed-tubers did not improve yield. The effect of the Monceren-Combi treatments was expressed by a decrease in the percentage of infected tubers, and by a reduction in the severity of black scurf infection. The material was most effective when applied in the planting bed at a concentration of 5 kg/ha. The next most effective treatment involved dusting and application in the planting strip with a concentration of 2.5 kg/ha, while the least effective was dipping the tubers in 2% Monceren-Combi. In conclusion, application of Monceren-Combi in the planting bed at a concentration of 5 kg/ha led to an increase in yield and substantial improvement in quality even when the mother tubers had not been disinfected.

Canon, a K-P Fertilizer Which Also Controls Plant Diseases

E. Barak Luxembourg Chemicals & Agriculture Ltd., Tel Aviv 61000, Israel [Fax: +972-3-5100474]

Canon is an aqueous solution which contains the salt potassium phosphite as the active ingredient. It is absorbed effectively through the various parts of the plant: through the leaves by foliar spray application or through the roots when applied to the soil. The potassiu m cation, as well as the phosphite anion, are systemically translocated all over the plant and may be consumed as nutrients.

Phosphorous acid has been known for some time for its fungicidal effect against several plant diseases, such as downy mildew in cucumbers and vineyards and Phytophthora diseases in avocado and citrus. Its mode of action is either direct inhibition of the oxidative phosphorylation process in the sensitive fungi, or indirect activation of the plant's natural resistance to the specific Oomycetes diseases.

152 Meetings

The use of Canon as a fungicide is recommended strongly, because of its high efficacy against certain Oomycetes plant diseases (Phytophthora, Peronospora, Pythium, Albugo) found in various crops (avocado, citrus, cocoa bean, strawberries, vineyards, Cucurbitaceae, ornamentals, etc.), its safety to the environment and its nutritious properties. (L)

Integrated Control of Grape Powdery Mildew by Foliar Sprays of Potassium Phosphate

M. Reuveni, 1 Tirza Zehavi 2 and R. Reuveni 3 1Golan Research Institute, Univ. of Haifa, Qazrin 12900 [Fax: +972-6-961930]; 2Extension

Service, Ministry of Agricuhure, Zefat 13100; and 3Dept. of Plant Pathology, ARO, Newe Ya'ar Research Center, Haifa 31900, Israel

Foliar sprays of 0.4--1.0% solutions of monopotassium phosphate fertilizer (KHzPO4 plus Triton X-100), commercial systemic fungicides or sulfur inhibited development of powdery mildew fungus caused by Uncinula necator on fruit clusters of field-grown grapevines (cvs. 'Chardonnay' and 'Cabernet Sauvignon'). The fungicide-based treatments were more effective in controlling the disease than the phosphate. Alternating treatments of phosphate fertilizer with an appropriate systemic fungicide or with sulfur, however, enhanced the inhibitory effect against the mildew and was similar to that of the commercial treatment with systemic fungicides. Similar results were obtained in semi-commercial model plots. The alternative treatment, in which the number of fungicide applications was reduced by 50%, offers new possibilities for disease control and reduction of pesticide usage. Phosphate solutions were not phytotoxic to plant tissue and did not affect the size of berries or cluster weight, as compared wtih the commercial treatment. However, lower cluster weights were recorded for the control of non-treated plots, due to mildew infection on berries. Determination of juice quality in berries indicated similar values of pH, Brix (% sugar) and total acids, regardless of the various treatments. The inhibitory effectiveness of the potassium phosphate, as well as the rapid absorption by the plant tissues, the low cost and its comparative environmental safety, render it a useful 'biocompatible' fungicide for application in the field for integrated disease control. (L)

Use of Bicarbonate Salts to Reduce Decay Development in Harvested Fruits and Vegetables

E. Fallik, 1 Shoshana Grinberg 1 and O. Ziv 2 1Dept. of Postharvest Science of Fresh Produce [Fax: +972-3-9683622] and 2Dept. of Field Crops,

ARO, The Volcani Center, Bet Dagan 50250, Israel

The commercial life of red sweet pepper fruits after harvest is limited by the development of decay caused mainly by Alternaria alternata Ft. and Botrytis cinerea Pers. Fungal diseases in fruits are usually treated with synthetic chemical fungicides. However, in recent years, public concern regarding pesticide residues on food crops has rendered registration of new fungicides very difficult. Alternative strategies which are safe, effective and economical are needed for control of postharvest diseases.

Bicarbonate salts are widely used in the food industry and have broad-spectrum antimicrobial activity. Potassium bicarbonate (PBC) inhibited in vitro mycelial growth, spore germination and germ tube elongation of B. cinerea and A. alternata. Germ tube elongation was found to be more sensitive than mycelial growth and spore germination to elevated PBC concentrations, as measured by EC~os. The inhibitory effect of PBC on the two fungi was probably due to the reduction in fungal cell turgor pressure, which resulted in collapse and shrinkage of hyphae and spores, and consequent inability of the fungi to sporulate. The PBC action was fungistatic rather than fungicidal.


PBC was found to be more effective than sodium bicarbonate in reducing decay development on harvested sweet red pepper. A prestorage dip of red sweet pepper fruits for 2 rain in 1% or 2% PBC solutions reduced fruit decay to a commercially acceptable level (5-8%) during storage and marketing simulation. However, a longer dipping time (4 min), especially at a higher PBC concentration (3%), led to weight loss, decreased firmness and further decay. (L)

Control of Molds Attacking Stored Wheat Grain Using Oregano Essential Oil and Thymol, Its Major Constituent

N. Paster, 1 Mazal Menasherov, 1 U. Ravid 2 and Irena Katzir 2 1Dept. of Stored Products, ARO, The Volcani Center, Bet Dagan 50250 [Fax: +972-3-9604428];

and 2Dept. of Medicinal Plants and Spices, ARO, Newe Ya' ar Research Center, Haifa 31900, Israel

The efficacy of oregano essential oil and its major constituent, thymol, in controlling molds of moist wheat grain (18.5% moisture content) was studied on 35-cm-high grain columns. The materials were applied as fumigants (6 g/kg grain) using air-circulating pumps to ensure their even distribution throughout the columns. Grain columns without any treatment, or in which the treatment materials were applied without air circulation, served as the controls. Thymol was highly effective in preventing mold growth, and infestation levels in the column (following 30 days of circulation) were 1% and 4% for the top and the bottom grain layers, respectively. Levels of infestation in a column treated (by the same procedure) with oregano essential oil were 40% and 23% for the top and the bottom layers, respectively. In columns in which the materials were applied separately, without circulation, a low level of infestation was recorded for the top layer only (4% and 6% infestation for thymol and oregano essential oil, respectively), whereas 100% infestation was recorded in the bottom layer, for both materials. Grains from the untreated columns showed 100% infestation in the various grain layers. The absorbance of thymol to the grains was analyzed using gas chromatography. The analyses revealed that in the column with air circulation, the same amount of thymol was absorbed in grains located at the top and at the bottom of the columns, whereas in columns without circulation the material was found only in grains at the top. It is concluded that both of the materials were efficient in controlling molds infesting wheat grain and therefore their use as alternatives to chemicals should be considered. Trials are now in progress to assess the optimal dosages and circulation time needed. (L)

Using Extracts oflnula viscosa for Controlling Plant Diseases on Fresh and Dry Postharvest Products

O. Ziv Dept. of Field and Garden Crops, ARO, The Volcani Center,


Several tests were conducted to investigate the effect of an aqueous extract of Inula viscosa obtained by boiling. It was found that adding an extract of L viscosa to potato dextrose agar (PDA), caused a considerable delay in the development of various species of pathogenic fungi. In two of the fungi, Botrytis cinerea and Rhizopus stolonifer, sporulation was delayed up to 12 days after inoculation. A linear correlation was found between the concentration of the I. viscosa extract in PDA and the delay in fungus development.

Comparison of extracts of several parts of the plant (young leaves, old leaves, stem, roots and flowers) which were dried with hot air for 5 days prior to extraction, revealed that the greatest inhibition activity was exerted by the extract obtained from boiling mature leaves in water; an abstract obtained from young leaves was significantly less effective. With the extracts from roots and flowers, inhibition was insignificant.

154 Meetings

A 10-min dip of peanut seeds in a boiled extract reduced significantly the infection by R. stolonifer, B. cinerea, Aspergillus niger and Aspergillus flavus, in comparison with untreated seeds. I. viscosa extracts significantly inhibited seed germination of various crops, and development of R. stolonifer and B. cinerea on postharvest grapes and tomatoes. No significant differences were found among sampled L viscosa plants from 18 different locations in Israel.

Among 12 pathogenic fungi, R. stolonifer was the most susceptible to L viscosa extract, and thus the fungus was used for bioassays of the extract's activity. (L)

The Role of Helicoverpa (Heliothis) in Enhancing Sunflower Head Rot Disease and the Applicability of Its Suppression

D. Shtienberg, 1 D. Zohar, 2 J. Dreishpun, 2 Y. Hefer 2 and A. Lior 2 1Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250 [Fax: +972-3-9683543];

and 2Dept. of Field Crops, Extension Service, Ministry of Agriculture, Tel Aviv 61070, Israel

The importance of sunflower head rot, caused by Rhizopus arrhizus, has increased in recent years in Israel. The pathogen induces moist head rot and reduces yield quantity and quality. The heads are resistant to infection at early stages of their development, but when anthesis has occurred at the disk florets that are located in the center of the head, the heads become highly susceptible. Injury of the heads is a prerequisite for infection and injuries may result from hail (in northern countries), birds or insects. The role of Helicoverpa (Heliothis) armigera larvae in enhancing sunflower head rot, and the applicability of its suppression, were studied in a set of experiments during the years 1992-95.

In a survey of commercial fields, the incidence of head rot and of Helicoverpa damage was recorded. We also noted whether only one of the pests developed, or if they coincidentally infected the heads. Analysis of the results revealed that injury caused by H. armigera larvae was apparent on 92% of the heads that were rotted by R. arrhizus, but only 39% of the heads injured by H. armigera larvae were also rotted. In another experiment it was found that the larvae were capable of feeding on achenes only until the development stage at which the achenes' color began to change from white to gray. Based on these findings, it was hypothesized that control of H. armigera larvae between the crop growth stage at which anthesis had occurred at the disk florets that are located in the center of the head, and the stage at which the achenes began to change color, may reduce head rot incidence. This hypothesis was tested in a field experiment and from seven observations conducted in commercial fields. Application of the insecticide endosulfan (1.0 kg a.i./ha) during that growth period resulted in a significant reduction (59% on the average) in the incidence of sunflower head rot. Large-scale implementation of these findings requires further evaluation. (L)

Control ofAlternaria solani in Potato and Tomato under Organic Management with Compost Extracts

Leah Tsror (Lahkim), 1 R. Barak 1 and R. Rodman 2 ~ Dept. of Plant Pathology, ARO, Gilat Regional Experiment Station, M.P Negev 2, 85410 [Fax:

+972-7-92' "7]; and 2 lsrael Bio-Organic Agriculture Association, Tel Aviv 67011, Israel

Early blight of potato and tomato is caused by Alternaria solani. The fungus infects mainly old leaves, creating typical concentric lesions. Later the disease spreads to the entire plant, which eventually dries up and dies. As a result, the yield may be decreased by 50% in susceptible cultivars. In addition, the disease can occur on fruits and tubers, thereby affecting the quality of the yield.

Under organic agricultural management one cannot use synthetic chemical agents (except copper derivatives, and even their use may be eliminated in the near future). Compost is being used routinely in organic agriculture. It is known that high quality compost may affect soilborne plant pathogens (e.g. Rhizoctonia, Pythium, Sclerotium rolfsii). Water extracts of composts were also reported to


have the potential to reduce foliage diseases such as Peronospora in grape. Phytophthora in potato, and Botrytis in vegetables. The present study demonstrated a reduction in early blight in organically grown potato and tomato, under field conditions, by spraying compost extracts.

Field experiments were conducted in organic plots at the Gilat Experiment Station. Potato and tomato plants were inoculated with A. solani, and different compost extract preparations were applied and compared with Funguran (a copper hydroxide formulation, produced by Jewnin-Joffe Industry, Tel Aviv), which is the accepted copper agent used in organic agriculture. Control of A. solani in inoculated tomatoes was accomplished by spraying compost extracts; this method compared favorably with the control achieved by applying Funguran. Two-week-old compost extract was more efficient than one-week-old in reducing disease incidence and improving yield. In potatoes, the efficacy of this treatment was not so conclusive as in tomatoes. However, data indicate a reduction in disease incidence and improvement of yield. In the spring season, 3-week-old compost extract gave better control than 10-day-old extract. In autumn as well as in spring, extracts produced at a ratio of 1:10 (compost:water; v/v) were more efficient than a 1:5 ratio preparation.

Control of early blight in these and possibly other crops (such as carrot) may be achieved by spraying compost extracts, which would facilitate more profitable organic management. (P)

The Use of Aspire TM in Commercia l Packinghouses for Control of Postharvest Decay of Citrus

H. Katz, 1 E. Chalutz, 2 S. Droby, 2 Lea Cohen, 2 Batia Weiss, 2 A. Daus, 2 Batia Horev, 2 Laura Chalupovicz, 2 E. Yogev 3 and A. Shaknai 4

1Ecogen Israel Partnership, Jerusalem 91042 [Fax: +972-2-733265]; 2Dept. of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250; a Luxembourg Chemicals and

Agriculture Ltd., Tel Aviv 61000; and 4Tnuport Export Ltd., Tel Aviv 61200, Israel

The yeast biocontrol agent Candida oleophila, developed by Ecogen Israel Partnership, was recently registered under the name Aspire T M by Ecogen Inc.USA. It has been tested under semicommercial and commercial packinghouse conditions during the past 3 years. Over 150 experiments have been performed in the pilot packinghouse located at the ARO, The Volcani Center, and more than 12 experiments have been conducted in commercial plants in Israel, the USA and Spain. During this period Aspire T M consistently reduced the level of molds caused by Penicillium digitatum and P italicum by approximately 50% in comparison with the water control. When applied following a pre-wash of 0.35% SOPP (sodium orthophenylphenate tetrahydrate), the molds were reduced by 90% as compared with the water control; SOPP alone reduced mold by only 50%. Application of Aspire T M with 50-200 ppm of TBZ, or imazalil (5-10% of commercial levels), resulted in control at a level equivalent to that of the commercial treatment. In commercial trials, Aspire T M has proved to be effective against sour rot caused by Geotrichum candidum, whereas the commercial treatment enhanced sour rot infection. Aspire T M can continue to grow on the peel even after the application of wax containing commercial combinations of TBZ (thiabendazole), imazalil and Ridomil and hormones commonly used in postharvest control. (L)

AQ10, a Biofungicide for the Control of Powdery Mildew of Grapes

D. Beth-Din a, M. Berson 2 and A. Sztejnberg ~ 1Ecogen Israel Partnership, Jerusalem 91042 [Fax: +972-2-733265]; 2 Luxembourg Chemicals and

Agriculture Ltd., Tel Aviv 61000; and aDept, of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100, Israel

AQ10 is a biofungicide developed by Ecogen Israel Partnership, based on the hyperparasitic fungus Ampelomyces quisqualis. The isolate AQ10, discovered in the laboratory of Dr. A. Sztejnberg

156 Meetings

at the Faculty of Agriculture, Rehovot, was found to be particularly efficient as a biocontrol agent for the control of powdery mildew (PM) in various crops. AQ10 attacks PM disease and causes a substantial reduction in the sporulation and dispersion of the pathogen. The development of the biofungicide was conducted on two levels: (i) elaboration of an easy-to-use formulation with high biological activity and a long shelf life; and (ii) extensive field testing throughout the world to determine the efficacy of AQ10 over a wide range of crops, with the principal focus on grapes. In an experiment on PM of grapes in France, AQ10 was sprayed at 7-, 10- and 14-day intervals and disease was reduced by 91%, 98% and 80%, respectively. The chemical standard reduced disease by 95% in comparison with the untreated control, but was significantly less effective than AQ10 applied every 7 days. In experiments conducted on varieties 'Carignan' and 'Semillon' in Israel, a treatment consisting of two applications of Bayfidan (triadimenol) and Systhane (myclobutanil), followed by three applications of AQ10 (with 0.3% mineral oil) reduced disease by 95% compared with that of the untreated control, and by 90% compared with two applications of chemicals alone. This integrated AQ10 treatment did not differ significantly from the chemical standard consisting of five applications of sterol inhibitors. In another experiment conducted on Carignan in Israel, the efficiency of AQ10 in combination with 0.3% mineral oil was tested. This combination reduced disease level to significantly less than that achieved by either component alone, and reduced disease by 82% in comparison with the control alone. The results of the field trials confirm that AQ10 is effective when used alone, or tank-mixed with 0.3% mineral oil, as part of an integrated pest management program. We recommend giving the sprays in the late afternoon or evening, when humidity is high. (L)

Studies of Biological Control of Powdery Mildew in Cereals by the Hyperparasite Ampelomyces quisqualis

G. Mairovich, 1 J.B. Marder, 2 D. Shtienberg 3 and A. Sztejnberg 1 1Dept. of Plant Pathology and Microbiology and 2Dept. of Agricultural Botany, The Hebrew University of Jerusalem, Faculty of Agricuhure, Rehovot 76100 [Fax: +972-8-9466794]; and

3Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel

Wheat and barley are frequently infected by the powdery mildew (PM) disease caused by Erysiphe graminis (EG) which is distributed worldwide. Ampelomyces quisqualis (AQ) is a fungal hyperparasite on PM in different crops. Interactions among the cereal host, PM and its hyperparasite were studied by examining changes in ultrastructure, physiology, crop yield and quality.

Scanning electron micrographs showed spherical AQ pycnidia occupying the bulbous bases characteristic of EG conidiophores on cereals. AQ hyphae advance along the conidiophore, where more pycnidia develop. The pycnidia eventually rupture, releasing spores that reinoculate EG. Optical microscopy of field samples showed the presence in PM cleistothecia of AQ pycnidiospores, which may be important for field survival of the hyperparasite. Tunneling electron microscope sections showed AQ invasion of EG haustoria within host plant epidermal cells. In PM-diseased cereals, we observed cell plasmolysis and damaged chloroplast morphology; plants that had been treated with AQ were less affected, but many plastoglobuli appeared in the chloroplasts. Measurements of chlorophyll content and organization gave a similar picture. Relative to controls, chlorophyll content declined to 21% in diseased plants compared with 74% in AQ-treated plants. Fluorescence measurements indicated that whereas in control plants >80% of the basal emission is associated with active photosystem II, in mildewed plants this declined sharply (,-~25% for wheat and ~38% for barley). AQ-treated plants showed ,-~67% (both cereals) of basal fluorescence from active photosystem II.

In greenhouse trials to determine optimal conditions for the hyperparasite, AQ suppressed EG sporulation by up to 70%. In wheat PM, AQ hyperparasitization plateaued after 4 h at 100% humidity while barley PM required > 10 h. This difference should be taken into account during application of


the biofungicide. AQ treatment was assessed as a pest control system by examining wheat grain production. PM reduced yield to 9%, whileAQ restored yield to 93% of non-diseased controls. These promising results should be verified in comprehensive field trials. (L)

Antifungal Activity of Yeast Cell Wall Materials Against Postharvest Fungal Pathogens

S. Droby, E. Chalutz, Laura Chalupovicz, Lea Cohen, Batia Horev, Batia Weiss and Smadar Lischinsky

Dept. of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250, Israel [Fax: +972-3-9683622]

Scanning and transmission electron microscopy revealed extensive production of extracellular materials covering the cells of the yeast Pichia guilliermondii. In an attempt to study the role of these materials in the antagonistic activity of the yeast against postharvest pathogens, an extracellular material was isolated from cells of the yeast by washing with LiC1 followed by 3 days of dialysis to remove the salt. When tested in vitro, the isolated material inhibited spore germination and germ tube elongation of several postharvest pathogens. The calculated ECso values for inhibition indicated a differential response of the various fungi to the material. The ECho for spore germination of these pathogens was obtained at concentrations ranging from 38 #g/ml to 300 #g/ml. Lower concentrations (22-230 #g/ml) of the material were needed to inhibit germ tube elongation. Application of extracellular material to surface wounds of grapefruit resulted in a marked inhibition of infection by Penicillium digitatum. Complete inhibition of infection was already evident at a concentration of 500 #g/ml.

The effect of the extracellular material in preventing natural infection of wounded grapefruit was also tested. Only 1.5% infection developed in fruits treated with 200 #g/ml followed by 9 days of incubation at 20~ At concentrations of 50 and 100 #g/ml, the percentage of infected wounds was 44 and 8.8, respectively. Control, non-treated fruits developed 92.2% infected wounds after the same period of incubation. (L)

F: PHYSIOLOGY AND PATHOGEN RESISTANCE TO CHEMICALS; GENETICS

Evaluation of Biocontrol Activity of Epiphytic Yeasts of Citrus Fruit Against Green Mold Decay Caused by PeniciUium digitatum

Smadar Lischinsky, 1 S. Droby, 1 Shulamit Manulis, 2 Lea Cohen 1 and Batia Horev 1 1DeDt. of Postharvest Science of Fresh Produce [Fax: +972-3-9683622] and aDept, of Plant

Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel

This study was aimed at isolation and characterization of the epiphytic yeast population of citrus fruit antagonistic to green mold decay caused by Penicillium digitatum. Ability to grow on elevated concentrations of NaC1, glucose and a wide range of temperatures was used as the criterion for the initial selection. Of 202 yeast isolates, selected at random following plating of surfaces, washings and isolations from wounds, 64 grew at temperatures ranging from 5 to 43~ Of 64 isolates, approximately 60 were found to grow on potato dextrose agar amended with either 10% NaC1 or 50% glucose. Following an assay performed on wounded grapefruit to test biocontrol activity, 46 isolates exhibited 70% or more inhibition of infection. Among these antagonists, four yeast isolates were highly effective against P digitatum on grapefruit. Complete inhibition of infection of surface wounds resulted when those yeast isolates were applied at a concentration of l0 s cells/ml. RAPD- PCR analysis showed that the population of the 64 yeasts tested consisted mainly of three groups of isolates, suggesting the presence of only three different yeast species.

158 Meetings

Classical identification (based on ability to grow on various carbon sources) of representatives of the three groups showed only two yeast species: Pichia guilliermondii and Debaryomyces hansenii. (L)

The Use of nit Mutants to Investigate Population Diversity of Fusarium moniliforme

M. Galperin and Y. Levy Dept. of Life Sciences, Bar-llan University, Ramat Gan 52900, Israel [Fax: +972-3-5354133]

Fusarium moniliforme is a filamentous ascomycete fungus which is pathogenic to a variety of animals and plants. In corn (maize), E moniliforme is associated with root, stalk and ear rots, and causes serious crop losses. Infected plant tissues may in some cases remain asymptomatic. Isolates of E moniliforme were collected from different parts of plants with or without disease symptoms grown in soils at different locations and from seeds of different corn cultivars. Isolates did not evince significant morphological differences when grown in culture. Nitrate non-utilizing (nit) mutants were generated from isolates on chlorate medium and used for determination of genetic diversity within isolates from different locations and from seeds. Complementation tests showed that 186 isolates belonged to 64 vegetative compatibility groups (VCG). The high VCG diversity of the population may be due to the introduction of multiple VCGs or sexual reproduction and the segregation of multiple vegetative incompatibility loci. For determination of genetic diversity in individual corn plants, two to eight isolates were collected in each plant from each of three loci: the ear, stem and root. In all cases more than one VCG was detected in each plant. In most cases at least two isolates recovered from several parts of the plant were vegetatively compatible. This finding indicates the possibility of systemic infection by E moniliforme. In another set of experiments, corn seedlings were inoculated locally by nit mutants. These nit mutants were recovered in seedlings, nodes, cob and seed samples of mature plants with/without symptoms. Recovered isolates were paired with respective tester strains in a heterokaryou test. The results indicate that E monitiforme is translocated systemically, nit mutants have the same infection ability as wild-type isolates, but they are more easily identified. Results show that nit mutants of E moniliforme are useful for studying population dynamics and genetic variability. (P)

Vegetative Compatibility among Populations of Verticillium dahliae, V. tricorpus and V. nigrescens in Israel

Nadia Korolev, 1 Talma Katan 2 and J. Katan 2 1Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250 [Fax: +972-3-9683543];

and Z Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100, Israel

Vegetative compatibility has been studied among populations of three soilborne species of Verticillium in Israel, using nitrate-nonutilizing nit mutants. V. dahIiae is known as a strong pathogen with a wide host range, whereas V tricorpus and V. nigrescens are considered weak pathogens or saprophytes. Three vegetative compatibility groups (VCG) were identified among 388 strains of V. dahliae isolated from 11 host plants (including weeds) and soil from 38 sites in Israel. By using various tester strains, the isolates from Israel have been assigned tentatively to VCG1, VCG2 and VCG4 of the Ohio (OARDC) system. Isolates of VCG1 occur in the north and those of VCG4 in the south of Israel, whereas isolates of the minor VCG2 do not show such localization. The VCGs differ from one another in colony morphology, pathogenicity on some host plants (e.g. cotton, eggplant) and response to temperature. Optimal temperature for linear hyphal growth was 21-24~ for VCG1 and 24-27~ for VCG4; 14 isolates of VCG2 showed variation within this range, with greater overall similarity to VCG4.


Six VCGs were found among 17 isolates of V. tricorpus from potatoes and soil from three southern sites. Four VCGs were found among seven isolates of V. nigrescens. These results show an apparent greater diversity in these species relative to that observed in V. dahliae. (P)

Fungal Germination and Appressoria Formation Using Plant Surface Signals

M.A. Flaishman a,~ and EE. Kolattukudy 1 1Neurobiotechnology Center, The Ohio State University, Columbus, 01-143210, USA

[Fax: +1-614-2925379]; and 2Present address: Dept. of Plant Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel

Physical and chemical signals from the plant have been suggested to trigger fungal germination and appressorium formation. Recently the surface wax from avocado fruits was found to trigger germination and differentiation into appressorium in Colletotrichum gloeosporioides spores that normally infect avocado fruits. Pathogens such as Colletotrichum spp. are known to remain dormant on the surface layer until the fruit ripens, at which time the fungus infects the fruit and causes major damage. How the fungus times its infection to coincide with the ripening of the fruit has not been elucidated until now. We found that the fungus uses ethylene, the ripening hormone, as a cue to initiate infection. Ethylene signaling of germination and multiple appressoria formation first observed on a glass surface was found also on fruit. The ethylene signal provides a fail-safe mechanism for the pathogen to time its infection to coincide with the ripening of the host fruit. The ethylene effect on the fungi appears to be a plant-like response as it could be replaced by propylene and was inhibited by silver ions and 2,5-norbornodiene. It appears that the pathogens have a plant- like mechanism to perceive and respond to the ethylene signal.

Both avocado surface wax and ethylene induce appressorium formation and the phosphorylation of mainly 29 kDa and 43 kDa proteins. Calyculin A, a potent inhibitor of protein phosphatases, induced, in yeast extract, appressorium formation and the phosphorylation of the same 29 kDa and 43 kDa proteins as did ethylene and avocado wax. These results indicate that protein phosphorylation is involved in the induction of appressorium formation by the two host signals, ethylene and avocado wax. (L)

Ethylene Does Not Activate Lesion Growth of Quiescent Infections of Colletotrichum gloeosporioides in Avocado Fruits

D. Prusky, C. Wattad and l iana Kobiler Dept. of Postharvest Science of Fresh Produce, ARO, The Volcani Center, Bet Dagan 50250, Israel

[Fax: +972-3-9683622]

Colletotrichum gloeosporioides conidia attack avocado fruits during their growth and remain quiescent until the fruit is harvested. After harvest and ripening, the pathogen becomes active and develops, resulting in visual symptoms of decay. Ripening of avocado fruits involves a significant increase in fruit respiration and ethylene evolution. It was suggested in the past by other research groups that ethylene evolution serves as a 'signal' for appressoria proliferation and initiation of decay development following natural infections in citrus and tomato. In our work it was found that ethylene at 5 or 45 ppm enhanced spore germination and appressoria formation on avocado wax in vitro, but only 45 ppm enhanced appressoria proliferation. Similar effects were obtained when 45 ppm ethylene was applied to whole fruits of avocado cv. 'Fuerte' infected with C. gloeosporioides conidia. In spite of the morphological effects on conidia of C. gloeosporioides on the avocado fruit, ethylene did not trigger the initiation of decay symptoms at different maturity stages. Decay development on ethylene-treated fruits was always related to the presence of antifungal diene in the peel of ripening fruits. (L)

160 Meetings

Virulence Factors, Mating Type and Sensitivity to Fungicides in Phytophthora infestans in Israel

Y. Cohen, A. Baider, Zipora Reshit and Sonia Farkas Dept. of Life Sciences, Bar-llan University, Ramat Gan 52900, Israel [Fax: +972-3-5354133]

Most isolates of Phytophthora infestans collected in Israel during 1995 belonged to the A1 mating type, whereas in previous years, most isolates were A2. Only 15% of the isolates collected were fully resistant to metalaxyl, unlike previous years in which most isolates expressed full resistance to this fungicide. Approximately 42% of the isolates were sensitive and 43% were moderately resistant to metalaxyl. The appearance of moderately resistant phenotypes may indicate sexual reproduction of the fungus, because resistance to metalaxyl is known to be monogenic semi- dominant. All isolates were sensitive to the fungicide cymoxanil, but this sensitivity was very variable. The presence of virulence factors was determined using a set of 11 potato differentials each containing a single gene (R1-R11) for resistance to late blight. We found that all isolates were complex, viz., had 5-9 virulence factors. The most frequent phenotype (56%) was H, which had the factors 1, 3, 4, 7, 8, 10 and 11. This phenotype is very frequent in Europe and may have entered Israel via potato seed trade. Current data for isolates collected during January-March 1996 show full sensitivity to metalaxyl. (L)

This research was supported by Sandoz Agro, Switzerland.

Streptomycin Resistance of Erwinia amylovora in Israel and Occurrence of Blossom Blight in the Autumn

Shulamit Manulis, 1 D. Zutra, 1 D. Ga'ash, 2 Frieda Kleitman, 1 0 . Dror, 1 S. Elisha, 1 I. David, 2 Dalia Rav-David, 1 Miriam Zilberstaine, 3 Z. Herzog 3 and E. Shabi a

1Dept. of Plant Pathology [Fax: +972-3-9603543]; and 2Dept. of Fruit Trees, ARO, The Volcani Center, Bet Dagan 50250; and aDept, of Crop Protection, Extension Service, Ministry of

Agriculture, Tel Aviv 61070, Israel

Fire blight was first detected on pear trees in Israel in 1985. Streptomycin has been the preferred bactericide for controlling the disease since 1986. Streptomycin-resistant Erwinia amylovora strains were first isolated from a pear orchard in the south in 1991. During 1994 and the spring of 1995, 45 orchards of pear, apple, loquat and quince from all over the country were sampled for streptomycin resistance. E. amylovora strains were isolated from infected tissue (flowers, shoots, cankers) on CCT medium (the differential medium of Ishimaru & Klos) and CCT-Sm (CCT supplemented with 100 #g/ml streptomycin sulfate). Representative colonies were verified by polymerase chain reaction using specific primers. Resistant strains were isolated from eight sites in the Hadera region (central coastal plain) and from a few orchards in the Golan Heights and Upper Galilee.

Since streptomycin did not provide adequate control of fire blight during the spring of 1995, the occurrence of resistance was investigated in autumn by sampling flowers from the autumn bloom. In Israel the common pear cultivars can bloom also during that season. Flowers were sampled during October-November from 23 pear orchards which had suffered fire blight during the spring and examined for the presence of the pathogen on flowers and for streptomycin resistance. Samples with a high population of bacteria (106-107 cells/flower, 25 flowers/sample) were spread on CCT and CCT-Sm. In ten orchards from the Hadera region, the pathogen population was resistant. These results prove that E. amylovora may cause blossom blight in the autumn and that streptomycin resistance is well established in this pathogen's population in Israel. The mechanism of resistance was examined using the internal portion of the streptomycin-resistance gene (SMP3 probe). No hybridization with this probe was obtained. Streptomycin resistance could not be transferred by matings to streptomycin-sensitive strains, which suggests that the resistance may be chromosomal. (L) Phytoparasitica 24:2, 1996 161

Influence of the Genetic Background and Environmental Conditions on Powdery Mildew of Melons

R. Cohen, x Y. Burger, x Shoshana Shraiber, 1 Y. Elkind 2 and Eilit Levin 2 1Dept. of Vegetable Crops, ARO, Newe Ya'ar Research Center, Haifa 31900 [Fax:

+972-4-9836936]; and 2Dept. of Field and Vegetable Crops and Genetics, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100, Israel

Powdery mildew is a limiting factor in melons. Most of the melon hybrids grown in Israel are resistant to race 1 of Sphaerotheca fuliginea, but are susceptible to race 2. The out-of-season and greenhouse-growing melon area are expanding and these changes are accompanied by changes in the response to diseases, including the response to powdery mildew and the pathogen races involved.

Disease incidence and severity were evaluated in the summer and autumn growing seasons in the open field. Disease evaluation was conducted on 17 genotypes including two resistant parental lines, seven susceptible parental lines and ten hybrids obtained by crossing the former with the latter.

In the autumn, disease incidence was 100% in all the genotypes tested, i.e., disease symptoms were observed on all of the leaves sampled. In the summer, disease incidence was lower, ranging from 10% to 80% in different genotypes. Disease severity, expressed as the percentage of leaf area covered by mildew, was much lower in summer than in autumn. The level of susceptibility of the different parental lines affected the response of the hybrid, particularly in autumn.

The response of 'Arava' and 'Revigal' (C-8), hybrids resistant to race 1 of powdery mildew, and of their parents, was tested in an open plot and under shading net transmitting 50% of sunlight. Disease severity was affected by light intensity. Low disease severity (less than 1%) was observed in all plants grown in the open, while under the net, disease was observed in the hybrids and the parents which had been considered resistant. In general, powdery mildew severity in the hybrid was intermediate between the resistant and susceptible parents.

The results indicate the need for a different approach to powdery mildew resistance breeding for out-of-season and greenhouse-grown melons. To increase the ability of the plant to overcome the disease under out-of-season conditions, both parents should carry resistance to powdery mildew, or at least very susceptible parents should not be used. (L)

Involvement of Nutrients and Plant Growth Regulators in Increased Growth Response of Tomatoes in Solarized Soils

J.M. Gri.inzweig, 1 H.D. Rabinowitch, 1 Y. Ben-Tal, 2 Maria Wodner 2 [deceased] and J. Katan 3

1Dept. of Field Crops, Vegetables and Genetics [Fax: +972-8-9468265] and aDept, of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot

76100; and 21nst. of Horticulture, ARO, The Volcani Center, Bet Dagan 50250, Israel

Soil solarization, a non-chemical and envi~oamentally sustainable method of soil disinfestation, promotes plant growth even in the absence of major pathogens; this phenomenon is termed Increased Growth Response (IGR). Leaves of tomato plants, 14 days after transplanting into solarized soil under controlled conditions, gained up to 122% more dry matter than leaves of control plants in non- solarized, pathogen-free soil, under controlled conditions. However, soil solarization did not affect' root dry matter, surface area or pressure. Therefore, IGR of tomato leaves is not initiated by a larger root system. It may result from higher N, P, Ca, Fe, Cu, Zn and Mn concentrations translocated from roots to shoot by xylem sap, with consequent higher concentrations in the leaves. Leaves of plants in solarized soit maintain high carbohydrate production over a prolonged period of time compared with control. This delay in leaf senescence may result from higher nutrient concentrations or increased levels of plant growth regulators. Two biosynthetic pathways for gibberellins were detected in tomato

162 Meetings

leaves using GC-MS: the early-C13-hydroxylation pathway leading to the biologically active GA1 and the non-C13-hydroxylation pathway leading to the active GA4. In addition, the active GA3 was identified. The endogenous concentrations of GA1 and GAz as measured by GC-SIM (selective ion monitoring) were up to 80% and 390%, respectively, higher in leaves from the solar treatment than in control. In addition, exogenous treatment with GA3 to the apex of plants in non-solarized soil resulted in improved growth, thus reducing differences between solarized and non-solarized soil from 30% to 7% for leaf fresh matter. Total cytokinin concentrations in xylem sap as evaluated by radio immunoassay were similar in the two treatments. These results show involvement of nutrients and plant growth regulators like gibberellins in IGR of tomatoes. Studies of cytokinin and auxin levels in tomato leaves and roots are in progress. (L)

G: INDUCED RESISTANCE TO PATHOGENS IN PLANTS; PHYTOALEXINS

Induced Resistance to Mal Secco Disease in 'Etrog' Citron and 'Rangpur' Lime by Infection by the Citrus Exocortis Viroid

Z. Solel, 1 N. Mogilner, 2 Miriam Kimchi, 1 R. Gafni 2 and M. Bar-Joseph 2 1Dept. of Plant Pathology [Fax: +972-3-9683543] and 2The S. Tolkowsky Laboratory, Dept. of

Virology, ARO, The Volcani Center, Bet Dagan 50250, Israel

Mal secco, caused by the fungus Phoma tracheiphila, is a severe vascular disease of various citrus cultivars. After penetration via wounds, the pathogen colonizes the xylem elements of the leaf, and progresses systemically in them into the branch wood, causing withering and dieback of branches. Field observations have indicated that nucellar lines of lemon, free of citrus exocortis viroids (CEVd), were more susceptible to mal secco than 'old lines' of the same varieties. This study was undertaken to examine our hypothesis that infection by CEVd induces resistance to mal secco.

'Etrog' citron and 'Rangpur' lime plants were inoculated with four isolates of CEVds and incubated for one year, during which the viroids spread systemically. The CEVds caused severe leaf curling and short internodes in the citron, but not in the lime. After leaf inoculation with P. tracheiphila, typical mal secco disease symptoms developed on leaves of both varieties, irrespective of prior infection by CEVd, but growth of the mycelium from the infected leaves into the branches was greatly affected by CEVd infection. In the citron, all CEVd isolates prevented mycelium growth into branches very effectively, whereas in the lime the isolates Sm-225 and Ex-76 were more effective than P3 and Y. (L)

Phytoalexin Production by Cypress in Response to Infection by Diplodia pinea f.sp. cupressi

Z. Madar, 1 M. Kimchi, 2 Z. Solel, 2 J. Riov 3 and A. Sztejnberg 3 1Dept. of Plant Protection, Keren Kayemet Le-lsrael, Qiryat Hayyim 26103 [Fax: +972-4-411971];

Z Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250; and 3Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem,

Faculty of Agriculture, Rehovot 76100, Israel

The antifungal terpenoid compounds cupressotropolone A (6-isopropyl-tropolone/3-glucoside) and B [5-(3-hydroxy-3-methyl-trans-l-butenyl)-6-isopropyl-tropolone /3-glucoside] were isolated for the first time from the bark of Italian cypress, after inoculation with the fungus Diplodia pinea f.sp. cupressi. The terpenoid compounds inhibited in vitro spore germination of D. pinea f.sp. cupressi, Seiridium cardinale, Alternaria ahernata and Verticillium dahliae. Production of the antifungal terpenoid compounds was also induced by S. cardinale, causing cankers on Italian cypress. The antifungal terpenoid compounds were fungistatic. Predisposition of cypress saplings


to extreme water stress enhanced the development of cankers caused by D. pinea f.sp. cupressi and enhanced fungal colonization in the bark and xylem in comparison with normally watered plants. Production of both cupressotropolone A and B in the bark, which commenced soon after the inoculation, was lower in plants predisposed to water stress. In plants treated with (aminooxy)acetic acid, a metabolic inhibitor, the cankers were significantly longer and the content of phytoalexins was significantly lower than in untreated plants. An extract of the bark of initiated cankers with 15% aqueous methanol, enhanced the subsequent development of the cankers, and reduced the content of both phytoalexins in the bark significantly in comparison with untreated plants. These results suggest that the terpenoid phytoalexins greatly inhibited canker development. (L)

H: MOLECULAR BIOLOGY

Differential Expression of Class C Protein Kinases during Early Development of Neurospora crassa

S. Oved and O. Yarden Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of


Conidial germination and hyphal elongation are common and fundamental events in filamentous fungal development and in many instances are essential phases of fungal infection and dissemination. Many developmental processes in fungi have been found to be elicited and controlled by various external and environmental signals; thus, the reacting cells are obliged to possess signal reception and transduction pathways. Protein phosphorylation has been clearly demonstrated to play a key role in the regulation of a wide variety of events in eukaryotic cells. The activity of class C protein kinases (PKCs) had been linked with one of the essential stages of signal transduction by external stimuli. As part of our strategy to study the control of development in filamentous fungi, we searched and analyzed the expression of proteins related to the PKC family in different developmental stages of the filamentous ascomycete Neurospora crassa. Immunochemical assays conducted with antibodies raised against several different PKCs showed that such polypeptides are present in N. crassa. Putative conventional as well as novel PKCs were identified. Two of the antibodies tested identified differential activity expressed by elevated PKC quantity during conidial germination. Using a pair of degenerate oligonucleotide primers designed on the basis of a highly conserved amino acid region in the PKC catalytic domain, and N. crassa genomic DNA as template, we were able to amplify (using low stringency polymerase chain reaction) PKC DNA gene fragments. One of the isolated fragments which was determined by sequencing to bear a close resemblance to PKC-encoding genes, was used as a probe to screen a N. crassa cDNA library. A positive 2.8 kb cDNA clone was isolated and the sequence of the clone determined as a gene encoding a novel PKC. The gene (designated pkc-2) was mapped to the left arm of linkage group IV between the mtr andfsr-51 genes. A 4.3 kb pkc- 2 transcript was observed in Northern blots. Transcripts are present in dormant spores, yet pkc-2 mRNA levels increase during spore germination. Additional genetic and biochemical analyses are being carried out in order to determine the possible role ofpkc-2 in N. crassa development. (P)

Inactivation of the Genes Encoding for Indole-3-Acetic Acid and Cytokinin Production Affects the Pathogenicity of Erwinia herbicola pv. gypsophilae

Anat Haviv, 1 Shulamit Manulis 2 and I. Barash 1 1Dept. of Botany, Tel-Aviv University, Tel Aviv 69978 [Fax." +972-3-6409380]; and 2Dept. of Plant

Pathology, ARO, The Volcani Center, Bet Dagan 50250, Israel

Erwinia herbicola ( E.h.) pv. gypsophilae induces gall formation on Gypsophila paniculata. Our previous work has demonstrated that pathogenicity of E.h. is associated with a plasmid (pPATH)

164 Meetings

which is present exclusively in all pathogenic strains. Furthermore, it was found that all pathogenic and non-pathogenic isolates of E.h. produce indole-3-acetic acid (IAA) through the indole-3- pyruvate (IPVM) pathway, whereas only the pathogenic strains produce IAA also through the indole- 3-acetamide (IAM) route. In contrast to IAA, cytokinin production was found only in pathogenic strains. The genes encoding cytokinin (etz) and IAA through the IAM pathway (i.e., iaaM and iaaH) were characterized and found to constitute a cluster on the pPATH. The chromosomal gene specifying the enzyme indole-3-pyruvate decarboxylase (ipdB) of the IPVM pathway has been characterized also in a non-pathogenic E.h. strain by M. Brandl and S. Lindow. The objective of the present work was to determine how mutations in each, all or various combinations of the above mentioned genes affect hormone secretion in vitro and pathogenicity in the pathogenic strain E.h. 824-1. Results obtained indicated that mutations in ipdB and iaaM or iaaH which lead to inactivation of the IPVM and IAM pathways, respectively, did not affect IAA production in vitro. Inactivation of the IAM pathway caused substantial reduction in gall formation. However, in contrast to the IAM pathway, the IPVM pathway did not affect pathogenicity. Mutation in the etz prevented cytokinin production, and caused significant reduction in gall development. Simultaneous mutations in all the above mentioned genes resulted in reduction in gall size but did not eliminate gall initiation. (L)

Localization of Chitin Synthase in Membranous Vesicles (Chitosomes) in Neurospora crassa

J.H. Sietsma, 1 Adi Beth Din, 2 Vered Ziv, 2 K.A. Sjollema 1 and O. Yarden 2 1Dept. of Plant Biology and Groningen Biomolecular Sciences and Biotechnology Institute,

University of Groningen, Biological Center, N1-9715 NN Haren, The Netherlands; and 2Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of Agriculture,


Chitin synthase activity is the rate-limiting step in chitin production in the fungal cell. Polyclonal anti-chitin synthase antibodies raised against the Saccharomyces cerevisiae chitin synthase II (CHS2) gene product (over-expressed in E. coli) were used to identify and localize chitin synthase in the filamentous ascomycete Neurospora crassa. A single band of approximately 110 kDa was observed in Western blots of total protein extracts ofN. crassa probed with these antibodies. However, several additional bands were labeled when membrane fraction proteins (microsomes) were probed. The chitin synthase activity measured in the mixed membrane preparation of N. crassa was significantly inhibited by addition of immune serum. The level of inhibition was positively correlated with the amount of antibody added. Histo-immunochemical localization of chitin synthase confirmed that the polypeptide is compartmentalized in membranous vesicles which fit the characteristics of chitosomes (between 50 and 100 nm in diameter and enclosed within a membrane), which are highly abundant in the vicinity of the hyphal tip. TEM analysis did not reveal chitin synthase in the plasma membrane. However, dense labeling of membrane-associated chitin synthase was observed by light-microscopic analysis of N. crassa protoplasts and at young hyphal tips. (L)

Screening of Sphaerothecafuliginea Isolates with DNA Markers

Ronit Greenberg, 1 R. Cohen, 1 O. Yarden, 2 Shoshana Shreiber 1 and Nurit Katzir 1 1Dept. of Vegetable Crops, ARO, Newe Ya'ar Research Center, Haifa 31900 [Fax: +972-4-9836936]

and 2Dept. of Plant Pathology and Microbiology The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100, Israel

Powdery mildew in cucurbits is caused mainly by Sphaerotheca fuliginea. Three races of the pathogen have been identified throughout the world and in Israel. Race identification is conducted using differential melon cultivars carrying different genes for resistance to the disease. The large


distribution and the wide host range of the fungi indicate that the number of possible pathotypes is probably larger than the three races detected by the currently available biological assay. The aim of this study was to assess the diversity among S. fuliginea isolates, using molecular markers.

Twenty-six isolates of S.fuliginea were collected from various locations and hosts in Israel. Race identification of each isolate was performed using the melon differential plants PMR 45 and PMR 6. DNA was isolated from conidia by a micro-isolation procedure and subsequently subjected to RAPD or inter-SSR PCR analysis. 244 10-mer arbitrary primers were applied in RAPD analysis and 20 of them demonstrated polymorphism between isolates. 39 SSR primers, 18 bp each, were applied in inter-SSR PCR; seven of them were polymorphic. Band sharing of the polymorphic products obtained with both methods will be analyzed to estimate the similarity among the isolates.

Fatty acids will be extracted from S. futiginea isolates and identified using gas chromatography. The data obtained will be compared with the molecular data in order to achieve a better understanding of S. fuliginea diversity. (L)

DNA Polymorphisms in Fusarium oxysporum f.sp. lycopersici

Sara Kalman, 1'2 Talma Katan 2 and D. Zamir 1 aDept, of Field Crops and Genetics, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76100 [Fax: +972-8-9468265]; and 2Dept. of Plant Pathology, ARO, The Volcani Center,

Bet Dagan 50250, Israel

A collection of 27 isolates of the tomato wilt pathogen Fusarium oxysporum f.sp. lycopersici (Fol) was used to estimate the genetic diversity among strains representing two physiological races (race 1 and race 2), two vegetative-compatibility groups (VCG 0030 and VCG 0031), and various geographic origins (Israel, Belgium, Greece, Italy, the Netherlands, South Africa, New Zealand, and California, Florida, Louisiana and Ohio in the USA). Isolates of Eo. radicis-lycopersici (Forl, the incitant of tomato crown-and-root rot) and Eo. metonis (Fom, the muskmelon wilt pathogen) were included for comparison.

DNA polymorphisms were studied by two approaches: (i) Fifteen genomic clones (FG-4, -25, -30, -34, -43, -103, -155, -163, -177, -213, -236, -261, -278, -281, -302) from Fol isolate FRC-0- 1078 (VCG 0030, race 2) served as probes, and along with three restriction enzymes (Eco RI, Eco RV, Dra I) were used to determine restriction fragment length polymorphism (RFLP). Ten RFLP probes differentiated between VCG 0030 and VCG 0031 of Fol, and two of them had no homologous sequences in VCG 0031. Some restriction-enzyme/probe combinations generated polymorphisms capable of distinguishing among Fol, Forl and Fom. (ii) The polymerase chain reaction (PCR) with nine 10-mer primers (OPL 1-4, 7, 10-13) was used to generate randomly amplified polymorphic DNA (RAPD) patterns. PCR products with primers OPL-2, -4, -7 and -11 enabled distinction between VCG 0030 and VCG 0031. Some primers, e.g. OPL-3, enabled partial distinction among Fol, Forl and Fom.

In cluster analysis, all Fol isolates grouped according to VCG. Neither RFLP nor RAPD resulted in groupings based on physiological races or geographic origin of Fol isolates, thus providing further evidence that VCG is an indicator of genetic relatedness among strains. (P)

Genetic Transformation in the Wheat Pathogen Septoria tritici

Smadar Pnini-Cohen, 1 Aviah Zilberstein, 1 Silvia Schuster, x A. Sharon 1 and Z. Eyal 1'2 1Dept. of Botany and 21nst. for Cereal Crops Improvement, The George S. Wise Faculty of Life

Sciences, Tel-Aviv University, Tel Aviv 69978, Israel [Fax: +972-3-6409380]

The fungal pathogen Mycosphaerella graminicola (anamorph Septoria tritici), the causal agent of septoria blotch of wheat, can bring on severe losses in yields. Isolates express a range of virulence

166 Meetings

patterns in the interaction with differential cultivars. Physiologic specialization is classified by quantification of symptoms (necrosis and pycnidia).

Suppression of pycnidia coverage was recorded in plants inoculated with isolate mixtures and following challenge inoculation. A transformation method for S. tritici was developed which enables the investigation of events associated with the interaction between isolates and wheat cultivars. The method uses fungal protoplasts in the presence of PEG (polyethylene glycol). The selectable marker gene hph - which confers resistance to hygromycin B, and the reporter gene uidA - which encodes the enzyme/3-glucuronidase, were stably introduced by co-transformation into the fungal genome of ~solates ISR398 and ISR8036, which differ in their virulence. Transformants were assessed for pycnidial coverage on seedlings of the differential varieties 'Seri 82' and 'Shafir'. The majority of the sub-isolates retained the virulence of the wild-type isolate, yet a few clones were identified with altered virulence. The genetic and molecular events associated with these S. tritici mutants are being investigated. The constitutive expression of uidA enables the elucidation of post-penetration events and the quantification of fungal biomass in planta. (L)

Induction and Primary Characterization of/%l,3-Exoglucanase from the Biocontrol Agent Ampelomyces quisqualis

Y. Rotem, O. Yarden and A. Sztejnberg Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of


Ampelomyces quisqualis, a fungus used as a biocontrol agent of powdery mildews, produces large amounts of the extracellular enzyme /3-1,3-exoglucanase in culture. The measured /3-1,3- exoglucanase activity of A. quisqualis culture filtrate was more than 100-fold larger than the enzymatic activity in filtrates of Neurospora crassa and Gliocladium roseum. The optimal activity of this enzyme is at pH 5.

Partial purification of the enzyme was carried out by anion exchange chromatography, preparative isoelectric focusing and SDS-PAGE. The protein purification process was monitored by enzyme activity assays at all stages, fl-l,3-exoglucanase has an estimated molecular mass of 117 and 60 kDa as determined by SDS-PAGE and gel filtration, respectively. This finding suggests the presence of an exoglucanase complex under certain experimental conditions. The isoelectric point of the enzyme is 6.1. Addition of dried fungal mycelium or pure laminarin (fl-1,3-glucan) to the growth medium induced enzyme production; this induction was not repressed by glucose.

In order to isolate the A. quisqualis fl-l,3-exoglucanase gene, a Cochliobolus carbonum cDNA clone, encoding such an enzyme, is being used as a heterologous probe. A unique fragment, which reacted positively to the probe, was observed in Southern blot analysis of A. quisqualis genomic DNA. As a primary step toward isolation of the glucanase gene, a cDNA library from A. quisqualis was prepared. The library was constructed from mRNA isolated from a growth phase which showed considerable /3-1,3-glucanase activity. The cDNA library is currently being screened with the heterologous probe. (L)

DNA Transformations and Molecular Analysis of the Chitin Synthase Gene (chsA) from the Mycoparasite Ampelomyces quisqualis

Nava Weiss, A. Sztejnberg and O. Yarden Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of


A first gene for chitin synthase was isolated from the parasitic fungus Ampelomyces quisqual&. Degenerate oligodeoxyribonucleotide primers, designed on the basis of conserved regions of the


chitin synthase gene family, were used to amplify a fragment of the A. quisqualis chsA gene. Subsequently, the polymerase chain reaction product was used as a probe in order to identify and isolate genomic clones harboring the entire chsA gene. chsA is 2786 nucleotides long, has one intron and encodes a 910-amino-acid polypeptide belonging to the class I chitin synthases. TATA (thymine-adenine-thymine-adenine), CAAT (cytosine-adenine-adenine-thymine) and a potential polyadenylation signal were identified in the non-coding regions of the gene. The chsA codon preference was found to be very similar to that determined for Neurospora crassa genes, with a high bias for GC (guanine-cytosine) at the third position of many codons. Low-stringency Southern hybridizations to A. quisqualis genomic DNA provided evidence for the presence of additional DNA fragments resembling chsA in the fungal genome, suggesting the presence of a'rnultigene family of chitin synthases in A. quisqualis. Protoplasts were prepared from the mycelium of A. quisqualis using a lytic enzyme, and a suitable osmotic stabilizer was found. They were used to carry out transformations of the hph gene, which can confer resistance to hygromycin B. Southern analysis showed evidence for the presence of the foreign DNA in the transformed cells. (P)

Detection of Aflatoxigenic Molds in Grains by Polymerase Chain Reaction

R. Shapira, 1 N. Paster, z Osnat Eyal, 1 Mazal Menasherov, 2 Anyet Matt I and R. Salomon 3 1Dept. of Biochemistry, Food Science and Nutrition, The Hebrew University of Jerusalem,

Faculty of Agricuhure, Rehovot 76100 [Fax: +972-8-9476189]; 2Dept. of Stored Products and 3Dept. of Virology, ARO, The Volcani Center, Bet Dagan 50250, Israel

Aflatoxins are carcinogenic metabolites produced by several members of the Aspergillusflavus group in grains and foods. Three genes: ver-1, apa-2, and omt-1, coding for key enzymes in aflatoxin biosynthesis, have been identified and their DNA sequences published. In the present study, three primer pairs were generated, each complementing the coding portion of one of the genes. DNA extracted from mycelia of five Aspergillus species, four Penicillium species and two Fusarium species were used as polymerase chain reaction (PCR) templates for each of the primer pairs. DNA extracted from peanut, corn and three insect species commonly found in stored grains was also tested. Positive results (DNA amplification) were achieved only with DNA of the aflatoxigenic molds A. parasiticus and A. flavus in all three primer pairs.

The detection limit of the PCR was determined using the primer pairs complementing the omt-1 and ver-1 genes. Sterile corn flour was inoculated separately with six different molds, each at several spore concentrations. Positive results were obtained only following a 24-h incubation in enriched media, with extracts of corn inoculated with A. parasiticus or A. flavus, even at the lowest spore concentration applied (10 z spores/g). No DNA amplification was observed from corn inoculated with other molds, even at the highest inoculum level (106 spores/g). It is concluded that genes involved in the aflatoxin biosynthetic pathway may form the basis for an accurate, sensitive and specific detection system, using PCR, for aflatoxigenic strains in grains and foods. (L)

I: NEW AND ESTABLISHED FUNGICIDES

Folpan - An Old Product With a New Use in Israel

O. Shifer Makhteshim Chemical Works Ltd., Be'er Sheva 84100, Israel [Fax: +972-7-280304]

Folpan (active ingredient, folpet) is produced exclusively by Makhteshim Chemical Works Ltd. and is marketed worldwide in many different formulations. Folpet, which belongs to the phthalimide group, is effective in preventing fungal diseases and is used throughout the world against a wide range of diseases in a variety of crops, e.g. downy mildew (Peronospora spp.) in a number of crops,

168 Meetings

the most prominent being grapevines; scab in apple; Botrytis in several crops; Pythium, Alternaria, and others. This product is used also as a seed treatment, as well as to prevent mold on the walls of houses. Folpet has a very low acute toxicity, with an oral LDso in rats >10,000 mg/kg, and is non-toxic to fish and bees.

In December 1993 the wettable powder formulation was granted registration in Israel for use on apple and pear for the control of scab, on almonds against Anthracnose, and on cucumber against downy mildew. One year later the water-dispersible granule formulation (WG) was registered for use on apple and pear, and in July 1995 for use on potato and carrot against Alternaria, with a note stating that the product has a side effect against potato blight, Phytophthora on potato, and powdery mildew on carrot. The registration for use on potato and carrot represents the culmination of experiments which were conducted over a 2-year period.

Alternaria on carrot is caused by the fungus Alternaria dauci, and is likely to cause destruction of the foliage and, as a result, may preclude machine harvesting and cause a reduction in yield. On the basis of experiments conducted on several farms in the Negev between 1993 and 1995, the dosage of 2.5 kg/ha plus 0.5% sticker was found efficient in preventing Alternaria and was safe to the crop. Folpan prevented Alternaria solani and Phytophthora infestans in potatoes at a dosage of 250 g/1000 m 2 plus 0.5% sticker and when applied at the proper time gave better control than a systemic fungicide.

Folpan has an important role in the range of products which are available for controlling AIternaria in crops such as potatoes and carrots, and can be used in alternation with dithiocarbamates (e.g. mancozeb). (L)

Stroby (Kresoxim-methyl) - A New Strobilurin Fungicide

E. Shabi, 1 E. Rosenberg, 2 Ester Hadar, a Y. Gottlieb, 3 T. Sando 4 and Tsilla Ben-David 5 1Dept. of Plant Pathology, ARO, The Volcani Center, Bet Dagan 50250 [Fax: +972-3-9603453]; 2Agan Chemical Manufacturers, Ashdod 77102; 3'4'S Extension Service, Ministry of Agriculture:

3 Rehovot 76325; 4 Ra'ananna 43100; and 5 Hadera 38364, Israel

Stroby is a fungicide belonging to a new chemical class, the strobilurins. These fungicides, which are analogs of strobilurin A - which was originally isolated from the fungus Strobilurus tenacellus (Pers. ex Fries) Singer, are active against fungi by inhibiting mitochondrial respiration. Stroby 50% WG (water-dispersible granules) (kresoxim-methyl, BAS 490F), a synthetic analog of strobilurin A, has been tested in Israel since 1994 and found to be very effective, without any toxicity to any crop, for the control of the following diseases: apple scab (Venturia inaequalis), apple powdery mildew (Podosphaera leucotricha), and powdery mildew of grapes (Uncinula necator), roses (Sphaerotheca pannosa), aster (Erysiphe cichoracearum), solidago (Oidium spp.) and strawberry ( Sphaerotheca macularis).

Stroby at 0.015% effectively controlled powdery mildew and scab in cv. 'Anna' apples in the coastal plain, and in cv. 'Top Red' (sensitive to scab) and cv. 'Jonathan' (sensitive to powdery mildew) in the Golan Heights; and at 0.02% it was very effective in controlling powdery mildew of wine grapes (cv. 'Carignan') and table grapes (cv. 'Superior'). At application rates of 0.02-0.04%, Stroby effectively controlled powdery mildew in a number of rose cultivars in 1994 and 1995; and of solidago in 1995. In that year Stroby 0.025% effectively controlled powdery mildew of asters and prevented infection as well. In spring of 1995, Stroby at 0.4 kg/ha gave good powdery mildew control of strawberries when the initial infection level was 13% coverage of leaves with spores and mycelia: it prevented new infection as well as reducing the initial infection level. Stroby at 0.8 kg/ha was insufficiently effective for control of oidiopsis (Leveillula taurica) in peppers.

In conclusion, Stroby is very effective for the control of apple scab and powdery mildew of various crops and has a unique and novel mode of action, different from that of currently used fungicides. Successive applications of currently used fungicides, with similar modes of action, have


promoted fungicide resistance of plant pathogens. The introduction of Stroby in disease management programs, in alternation or combination with currently used fungicides, may delay or prevent the development of resistance to fungicides by plant pathogens. (L)

D o m a r k - A New Fungicide

M. Berson 1 and the Agrochemical Team, 1 and Esther Hadar 2 1Luxembourg Chemicals and Agriculture Ltd., Tel Aviv 61000 [Fax: +972-3-5100474];

and 2Extension Service, Ministry of Agricuhure, Rehovot 76324, Israel

Domark (tetraconazole) is a triazole fungicide produced by Isagro Ricerca, Italy. The product inhibits the synthesis of ergosterol in phytopathogenic fungi and is intended for the control of powdery mildews, rusts, scab, Cercospora spp. and other diseases. It has good systemic efficacy and the distinct advantage of movement in woody plants. Domark is available as a 10% E.C. of the a.i., tetraconazole. The diluted formulation is sprayed on the canopy in most stages of plant growth, at 7-10-day intervals, depending on the intensity of the disease. During the last 2 years we have examined the efficiency of Domark in controlling powdery mildew in various crops. At a rate of 300--400 ml/ha (or a concentration of 0.03-0.04% in orchards), the efficiency of the product was excellent. In Cucurbitaceae (watermelon, zucchini and cucumber), 70-100% control of powdery mildews was achieved, significantly different from the untreated control and comparable to that of standard treatments, such as obtained with Anvil (hexaconazole), Systhane (myclobutanil), or Ophir (penconazole). In experiments conducted in vineyards with medium or high powdery mildew infestation, Domark gave excellent control and maintained disease-free bunches throughout the growing period. In cvs. 'Carignan' and 'Semillon', infestation in the control bunches reached approx. 90% vs 0.3% in the Domark treatment. In mango, Domark controlled powdery mildew on the inflorescences with especially high efficiency - 95-100% - which enabled a yield gain of 65% vs the untreated control. In similar trials on roses, the treatment was effective even when applied on the existent disease, with a 85-100% level of control vs untreated. The product excelled in controlling powdery mildew equally to the experimental product kresoxim-methyl (new chemical group) in cultures such as apples, roses and aster. Domark was active also in control of other diseases, such as rusts in fruit tree orchards and flowers, Cercospora in vegetables and peanuts, and scabs in fruit tree orchards.

In conclusion, Domark is a new fungicide in Israel, which excels in control of powdery mildews and improves our capability of keeping crops disease-free. Although it does not belong to a new chemical group, the results of our trials point up Domark's excellent efficacy in controlling powdery mildews. Its inclusion in the management scheme of powdery mildew control in various cultures, viz., vegetables, grapes, fruit trees and ornamentals, will help farmers in better controlling these diseases. (L)

J: VIRUSES AND COMBATTING VIRUS DISEASES

Viruses in Anemone

J. Cohen, 1 A. Franck, 1 N. Umiel, 2 E. Hadar, 3 S. Finkelstein 4 and A. Gera 1 1Dept. of Virology [Fax: +972-3-9604180] and 2Dept. of Ornamental Horticulture, ARO, The

Volcani Center, Bet Dagan 50250; 3Extension Service, Ministry of Agriculture, Rehovot 76324; and 4Extension Service, Ministry of Agriculture, Afula 18100, Israel

Anemone is a popular ornamental plant. Approximately half of the tubers are produced for flower production and half for gardening. Cultivation of anemones in Israel was discontinued in 1979 due to disease contamination of imported propagation stocks. In a breeding program conducted

170 Meetings

at the Department of Ornamental Horticulture, N. Umiel and his colleagues released two cultivars, 'Jerusalem' and 'Galilee', for commercial cultivation. Growing areas are expanding and the crop is becoming important. Israel produces a high quality propagation material for both local consumption and export.

Anemone is highly susceptible to viral diseases, with seven viruses infecting anemones having been detected worldwide. During surveys conducted in Israel in 1970 and repeated in 1995, the following viruses were detected: cucumber mosaic virus (CMV), turnip mosaic virus (TuMV) and tobacco necrosis virus (TNV). The first and the second are transmitted in a nonpersistent manner by aphids and the third is vectored by the zoospores of Olphidium brassicae. None of the three viruses is seed-transmissible. Recently, an unidentified disease, causing plant stunting and flower malformation of anemone, was observed. The disease can cause high losses. Identification of the causal agent is in progress.

The continuation and expansion of anemone export depends on the production of virus-free propagation material. A cultivation technique which will ensure the production of virus-free propagation material, was suggested. (L)

Detection of Turnip Mosaic Potyvirus in Ornamental Allium

A. Gera, 1 J. Cohen, 1 A. Franck, x S. Levy, 1 R. Salomon I and D.-E. Lesemann 2 1Dept. of Virology, ARO, The Volcani Center, Bet Dagan 50250, Israel [Fax: +972-3-9604180];

and 2 Inst. of Biochemistry and Plant Virology, Federal Research Institute for Agriculture and Forestry, Braunschweig 38104, Germany

The genus Allium is a member of the family Alliacea, which comprises more than 600 species. Recently many Allium species have been used for gardening and cut flowers as well as pot plants. Some species have become fashionable in international flower markets. In Israel three Allium species are cultivated on an industrial scale: A. aflatunense, A. sphaerocephalon and A. ampeloprasum. Species of the genus Allium are susceptible to various viral diseases. In garlic and onion, viruses with flexuous filamentous particles have been known worldwide for many years. Recently we reported the occurrence of onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV) in garlic (A. sativum) grown in commercial fields in Israel.

Since 1993 we have noted filamentous viruses in cultivated A. ampeloprasum grown in commercial greenhouses in Israel. An isolate of this virus was identified as a strain of turnip mosaic potyvirus (TuMV). Symptoms on infected plants include systemic chlorosis and yellow stripes accompanied by growth reduction. Leaves were distorted, often showing necrotic flecking. The virus was readily transmitted mechanically and in a non-persistent manner by aphids. Particles from crude extracts of infected Nicotiana benthamiana had a normal length of 806 nm. The A. ampeloprasum isolate was in particle length indistinguishable from a typical TuMV isolate which has a normal length of 793 nm. Cells of infected plants also contained pinwheel-shaped inclusion bodies that are typically induced by potyvirus infections. Polyacrylamide gel electrophoresis of purified denatured virus revealed a single polypeptide band, 35,000 daltons in size. The virus strongly reacted with antiserum to typical isolates of TuMV in immnnoelectron microscopy and Western blotting but not with antisera to several other potyviruses. Based on serological reactivity, particle morphology, aphid transmission and cytopathology, the virus was identified as an isolate of TuMV. (P)

Tolerance to Cucumber Mosaic Virus Derived from 'Perennial' Capsicum annuum and Analysis of Virus Level

M. Lapidot, 1 S. Cohen, 1 Rachel Ben-Joseph, x S. Ben Harush, 2 M. Pilowsky 3 and C. Shifriss 3

Phytoparasitica 24: 2, 1996 171

I Dept. of Virology [Fax: +972-3-9604180] and aDept, of Plant Genetics, ARO, The Volcani Center, Bet Dagan 50250; and 2 Hazera Seed Company, Qiryat Gat, Israel

By means of pedigree and backcrossing breeding procedures, tolerance to cucumber mosaic virus (CMV) was introduced from an Indian small-fruited hot pepper accession, 'Perennial') into several 'Bell'-type lines. The tolerance was found to be recessive with polygenic inheritance. Selection was performed based on symptoms appearance, following mechanical inoculation with CMV. Selection under greenhouse conditions was as effective as selection under field conditions. ELISA was used to follow virus levels in the upper systemic leaves in the several pepper lines. No correlation was found between virus accumulation levels and degree of tolerance to the virus, i.e., between symptom severity and fruit yield. Since the small Perennial fruits weigh only a few grams, it was necessary to perform over six backcrossing generations in order to arrive at the typical fruit weight of Bell peppers (150-250 g). Each backcross generation was followed by growing its F1 and F2 generations for screening to CMV tolerance. Since tolerance is recessive, a large number of lines are being bred as source material for the production of hybrid cultures.

The agronomic value of the tolerant lines and the variation in response to CMV were discussed. (L)

Development of Transgenic Tomato Cultivars Expressing Replicase-Mediated Immunity to Cucumber Mosaic Virus

A. Zelcer, 1 Dalia Wolf, 1 J.-E. Faure, 1 M. Pilowsky, 1 M. Bar, 1

S. Cohen, 2 A. Rosner 2 and A. Gal-On 2 1Dept. of Plant Genetics [Fax: +972-3-9669642] and 2Dept. of Virology, ARO,

The Volcani Center, Bet Dagan 50250, Israel

Cucumber mosaic virus (CMV) epidemics occur worldwide in open field tomatoes. The symptomatology varies from mild mosaic and 'shoestringlike' foliage, to necrosis and stunting. Yield is severely affected: fewer fruits, of small size and delayed maturity, are found on infected plants. At present, no resistant commercial cnltivars are available, since all known resistances are inconsistent, genetically complex or difficult to exploit due to hybrid sterility. The generation of transgenic resistance to CMV in tobacco by transformation with a defective replicase gene from this virus has been reported [Anderson et al., PNAS 89:8759]. We obtained from this research group the plasrnid pCMV N/B-23, which carries the defective gene, and used it in all experiments reported herein.

Transformation methods were adapted for tomato lines from our breeding collection. Three parental lines and two advanced breeding lines were transformed with Agrobacterium tumefaciens harboring the pCMV N/B-23 Ti-binary vector. A total of 139 transformants were isolated. Individual R0 plants were selfed in a greenhouse and R1 seeds were germinated in the presence of kanamycin, to discard non-transformed segregants. Kanamycin-resistant seedlings were mechanically inoculated with the CMV-Fny strain at high titers (0.5-1.0 mg/ml).

The response of R1 populations fitted into three different categories: (i) fully susceptible progeny (all seedlings exhibited symptoms); (ii) fully resistant progeny (all symptomless seedlings); and (iii) intermediate type progeny (mixture of seedlings with full symptoms and symptomless seedlings, at variable proportions). Symptomless plants from categories ii or iii were probed for CMV by ELISA or by inoculation onto appropriate differential hosts. In all instances plants exhibited an immunity response: no virus was ever detected in upper leaves.

CMV is normally transmitted under field conditions in a non-persistent fashion by many aphid species. We therefore mimicked field conditions by challenging resistant lines with viruliferous aphids. Our observations indicate that lines classified as totally immune by mechanical inoculations, show also immunity to aphid-mediated inoculations.

172 Meetings

Additional data on molecular characterization of transformants and on inoculation of resistant lines with different CMV strains were presented. (L)

Cross-Protection against Zucchini Yellow Mosaic Virus: Results of the 1995 Season

E. Lev, x H. Livne, 1 S. Omer, 2 H. Yunes, 2 S. Singer, 3 A. Gal-On 3 and B. Raccah 3 1Nahal Oz 85145 [Fax: +972-7-805407]; 2Extension Service, Ministry of Agriculture, Tel Aviv

61070; and aDept, of Virology, ARO, The Volcani Center, Bet Dagan 50250, Israel

During the 1995 season, an attempt was made to protect watermelon and melon fields in Israel from zucchini yellow mosaic virus (ZYMV) by cross-protecting the crops with a mild ZYMV strain (ZYMV-WK). Cross-protection was examined in five different geographical regions: northern Negev, southern coastal plain, western and upper Galilee, and the Golan Heights.

In the early spring season, experiments were carded out at Massu'ot Yizhaq in watermelon, and at Nahal Oz in melon; in the early summer, at Shomrat and Lohame haGeta'ot in watermelon; and in the late summer, at Yir'on and Yonatan in watermelon. In each of the watermelon fields protection was provided in one of two ways: frequent spraying with mineral oil, or cross-protection with the ZYMV-WK strain. The experimental scheme was similar at all sites: 0.1 ha was planted with protected seedlings in a commercial field that received mineral oil treatments. The seedlings were planted in the field one week after they had been inoculated in the nursery, and concomitantly leaf samples were taken at planting to determine the rate of infection with ZYMV-WK.

The effect of cross-protection on the yield was determined for watermelons by comparison with plots that were treated with mineral oils. In all the watermelon fields the yield in the cross-protected plots was not lower than in the oil-treated plots. However, in watermelons, infectivity with ZYMV was recorded only in the early spring, and therefore the benefit of cross-protection was noted only at Masuot Yizhaq (an additional yield of 1174 kg/0.1 ha).

At Nahal Oz cross-protection was examined in melon cvs. 'Rina', 'Ronit' and 'Ein-Dor'. Rina and Ronit were sown in six mini-plots (20 plants in each, equivalent to 20 mZ). The seedlings were inoculated in the field with ZYMV-WK 2 weeks after germination. The control plots were left untreated, and were heavily infected by severe ZYMV. The average yield in the protected plots was 997 kg/0.1 ha higher than in the untreated control. Cv. Ein-Dor was tested on a larger scale (0.1 ha), and the additional yield in the inoculated plots was 200 kg/0.1 ha.

The results of the 1995 experiments indicated that ZYMV-WK was harmless in the regions where it was tested. Moreover, in the early spring, when ZYMV infection was the most severe, there was a remarkable and significant yield increment compared with the uninoculated control. (L)

Breakdown of Resistance against Tomato Mosaic Virus (ToMV) in Pepper Varieties Grown in Greenhouses

Y. Ant ignus ) Malenia Pearlsman, 1 O. Lachman, 1 S. Cohen 1 and A. Koren 2 1Dept. of Virology, ARO, The Volcani Center, Bet Dagan 50250 [Fax: +972-3-9604180];

and 2 Hishtil Nurseries, Moshav Nehalim 49950, Israel

Sweet pepper seedlings (var. 'Mazurka') that were grown in greenhouses in Israel during August 1995 expressed disease symptoms that included necrotic lesions on the lower part of the stem, diffuse lesions on leaves, and sometime mosaic on apical leaves and distortion of the apical part of the plant. This syndrome led eventually to collapse of the plants. Later in the season it was possible to detect necrotic lesions on the stems of older plants, followed by their wilting. Significant economic damage occurred in some greenhouses where the disease had been found in the previous year. Electron microscope analysis of samples from diseased pepper plants indicated the presence of virus particles with a morphology characteristic of tobamoviruses. These samples reacted positively to antiserum


against tobacco mosaic virus (TMV). The virus that was isolated from pepper has a host range identical to that of ToMV. Moreover, when the pepper isolate and an identified tomato isolate of ToMV were inoculated to pepper (var. Mazurka) via the roots, both induced systemic necrosis in stems identical to the symptoms that were observed in the field.

In additional experiments it was shown that high temperature or high inoculum levels may lead to breakdown of resistance against ToMV in pepper varieties conferring the tm3 resistance gene. (L)

Use of the N-Terminal Segment of the Maize Dwarf Mosaic Virus Coat Protein as Competitor in the Virus Spread

R. Salomon 1 and Francoise Bernardi 2 1Dept. of �88 ARO, The Volcani Center, Bet Dagan 50250, Israel [Fax: +972-3-9604180];

and 21nst. Jacques Monod, 75251 Paris Cedex 05, France

Numerous investigations have revealed that for a successful potyvirus aphid transmission, intact virions and a viral coded protein, the helper component (HC-pro), are essential. Our previous work demonstrated that the whole N-terminal region of the coat protein (CP) is needed for aphid transmission. This CP segment is composed of approximately 60 amino acids (aa) residues, lacks a defined structure, and has some domains with biological functions. At a distance of 5 aa from the N terminus, there is a site of aspartic acid, alanine and glycine (DAG), which are required for aphid transmission. To study the functions of the various domains in this protein segment directly, we utilized specific nucleic acid primers for RT-PCR reactions. The complementary DNA obtained was cloned and expressed in bacteria. The bacterially expressed product was Maltose Binding Protein-N terminal CP fused protein (MBP-N ter CP). This protein was purified by affinity column chromatography and used in aphid transmission-competition experiments, by membrane feeding prior to acquisition of maize dwarf mosaic virus (MDMV) from infected corn plants. Proteolytic cleavage products of the fused protein were also utilized for competition, as control MBP alone was used. Only MBP-N ter CP or its proteolytic cleavage products inhibited almost completely aphid transmission of MDMV. Additional experiments were performed with a protein product from which the three aa domain DAG was removed. This modified protein did not compete in MDMV aphid transmission. Therefore we concluded from these results that the N ter CP of the potyviruses is also involved in the virus aphid acquisition step. (L)

Use of Reflective Mulches to Reduce Potato Virus Y Potyvirus Incidence in Seed Potato

C.D. DiFonzo, 1 D.W. Ragsdale 2 and E.B. Radcliffe 2 1Dept. of Virology, ARO, The Volcani Center, Bet Dagan 50250, Israel [Fax: +972-3-9604180];

and 2Dept. of Entomology, University of Minnesota, St. Paul, MN 55108, USA

Oat straw and white plastic mulches were tested for reducing aphid landing rates and potato virus Y (PVY) Potyvirus incidence in seed potato (Solanum tuberosum L.) in North Dakota (USA). Aphid landing rates were measured using green tile traps, and PVY incidence was determined by serological testing of tuber progeny. Mulches had a significant impact on aphid landing rates, with average number of aphids collected per week greater in plastic-mulched plots (38.3) than in straw-mulched (21.1) or unmulched (22.6) plots. Reported vectors of PVY constituted 93% of the 4,589 aphids collected during the season in tile traps. The five most common species captured were Lipaphis erysimi (55.2%), Rhopalosiphum padi (15.9%), Sitobion avenae (6.5%), Schizaphis graminum (5.3%) and Rhopalosiphum maidis (1.9%). PVY incidence was two times lower in straw- mulched plots (24.7%) than in plastic-mulched (49.4%) or unmulched (50.1%) plots. The reduction

174 Meetings

in PVY incidence in straw-mulched plots occurred in the inner rows. The effect of the straw mulch can be completely explained by a decrease in aphid landing rate. It was hypothesized that volunteer oat seedlings in these plots increased plant diversity and density, leading to a reduction in the number of viruliferous aphids reaching potato plants. Mulches may be impractical for use in potato, but undersowing potato plots with small grains may be a way to reduce PVY incidence. (L)

Defective RNAs in Plants Infected with Citrus Tristeza Virus

M. Mawassi, 1 Elzbieta Mietkiewska, 1 M.E. Hilf, 2 Lilach Ashulin, 1 A.V. Karasev, 3

R. Ga fny ] R.E Lee, 3 S.M. Garnsey, 2 W.O. Dawson z and M. Bar-Joseph 1 1 The S. Tolkowsky Laboratory, Dept. of Virology, ARO, The Volcani Center, Bet Dagan 50250, Israel [Fax: + 972-3-9604180]; 2 USDA -ARS, Horticultural Research Laboratory, Orlando, FL

33803, USA; and Z Citrus Research and Education Center, University of Florida, Lake Alfred, FL 33850, USA

Plants infected with several isolates of the citrus tristeza virus (CTV) were found to contain multiple species of RNA molecules with features similar to defective-interfering RNAs. The dsRNAs extracted from serial passages of the VT isolate showed considerable variation both in the presence and in the relative abundance of the defective-RNA (D-RNA) bands. The D-RNA molecules were found to be encapsidated in the CTV particles. Sequence analysis of the three VT D-RNA molecules of 2.4 kb, 2.7 kb and 4.5 kb, revealed that they were composed of two regions corresponding to 1151, 1818 and 4036 nucleotides from the 5' and to 1259, 938 and 442 nucleotides from the 3' termini of the CTV-VT genomic RNA, respectively. These were the first defective RNAs to be reported for a member of the closterovirus group. (L)

Native Citrus Tristeza Virus Virions are Heterodimers Consisting of Two Segments of ,,~2000 nm and ,-~85 nm, Separately Encapsidated by Subunits of Two Different

Coat Proteins

Lilach Ashulin, 1 M. Mawassi, a G. Yang, 1 A. Frank, 1 R. Gafny, 1 VJ. Febres, 2 C.L.

Niblett, 2 R.E Lee 3 and M. Bar-Joseph 1 1 The S. Tolkowsky Laboratory, Dept. of Virology, ARO, The Volcani Center, Bet Dagan 50250, Israel [Fax: +972-3-9604180]; 2plant Pathology Dept., University of Florida, Gainesville, FL

32611-0680, USA; and 3 Citrus Research and Education Center, University of Florida, Lake Alfred, FL 33850, USA

Immuno-electron microscopy (IEM) of 'dip' preparations from citrus tristeza virus (CTV)- infected plants using antibodies dCP or p27 against the CTV divergent coat protein showed decoration of a ~85 nm segment at one end of the particles. The dCP encapsidated segment was not decorated by antibodies against the viral coat protein. Both normal length (~2000 nm) and shorter particles which apparently are encapsidating defective (D)-RNA molecules, were found to show a decorated segment of a similar size. The decorated segment was not found in CTV particles, purified by the sucrose cesium-sulfate (SC) step gradient. Ultracentrifugation on a 10-30% phosphate sucrose (PS) step gradient, however, prevented the shearing of the tail segment. RNA extracts from CTV-VT particles isolated either by the SC or the PS step gradients were found to contain the 19.3 kb genomic and one or more defective(D)-RNAs of 2.4-4.5 kb. An additional RNA band of ,,~0.8- 0.9 kb was revealed in PS gradient fractions with native CTV particles. Additional experiments are in progress in order to test whether the dCP subunits encapsidate the ,-,0.8-0.9 kb RNA molecules.

The results confirm recent observations made by Agranovsky et al. [Proc. Natl. Acad. Sci. USA 92:2470-2473 (1995)] with the beet yellows closterovirus (BYV) which suggested that the


BYV coat protein does not encapsidate all parts of the native virion and that the remaining part was encapsidated by a different coat protein (th e diverged copy), resulting in an unusual heteromeric structure which they named 'rattlesnake'. (L)

Genomic Characterization of the VT Isolate of Citrus Tristeza Virus

M. Mawassi, Elzbieta Mietkiewska, Rose Gofman, G. Yang and M. Bar-Joseph The S. Tolkowsky Laboratory, Dept. of Virology, ARO, The Volcani Center, Bet Dagan 50250, Israel

[Fax: +972-3-9604180]

The complete 19277 nts sequence of the VT seedling yellows isolate of citrus tristeza virus (CTV) from Israel was characterized and found to have an identical genome organization to that of the previously determined CTV-T36 isolate from Florida, except that ORF 1 of CTV-VT was 66 nts shorter due to two widely separated 18 nt deletions. Sequence comparison of CTV-VT and CTV-T36 revealed approximately 85% identity throughout the ten 3' ORFs, but only 60--70% identity throughout ORF 1. The 5' nontranslated regions were only 60% identical, whereas the three nontranslated regions were 97% identical. The transition between regions of similarity and deviation was gradual, suggesting that the sequence similarities and differences compared to CTV- T36 were unlikely to have arisen from a recent recombination event between a close T-36 relative and a distantly related CTV isolate. (L)

K: NEMATODES

Isolation and Chacterization of the First Collagen Gene from the Root-Knot Nematode Meloidogyne javanica

Hinanit Koltai, 1 N. Chejanovsky, 2 B. Raccah 3 and Y. Spiegel 1 Depts. of 1Nematology, 2Entomology and 3 l~rology, ARO, The Volcani Center,


The root-knot nematode Meloidogyne javanica is a major pest of many crops. The use of traditional strategies to manage plant-parasitic nematodes is limited due to high costs, incomplete control, and lack of sufficient specificity. The entire surface of nematodes is covered by a multilayered cuticle, which consists mainly of collagen proteins. We propose to identify and characterize root-knot nematode cuticle collagen genes and their developmental expression, as a prelude to investigating the potential of managing plant-parasitic nematodes by targeting and disrupting their cuticles. This novel approach would involve no risk to non-target soil organisms or to human health.

We have identified, cloned and characterized the first cuticular collagen gene from the plant- parasitic nematode M. javanica, the gene mjcol-1. This gene putatively encodes a 32 kDa collagen protein, including a propeptide which possesses a subtilisin-like protease cleavage site. Sequence of mjcol-1 gene upstream (5') region revealed two regulatory elements. Downstream (3') to the gene a putative poly-A site was observed. Transcription of the gene was detected in the developing eggs and, to a lesser extent, in infective juveniles and adult females. The basic structure of the predicted protein sequence of the root-knot nematode cuticle collagen mjcol-1 was found to be primarily similar to the Caenorhabditis elegans dpy-7 gene, with 52% homology between the two gene sequences and up to 100% homology at the conserved regions. One of the non-conserved features of the mjcol-1 gene was found to be the number and position of the tyrosine residues. Since triple helical conformation is formed through covalent tyrosine-tyrosine bonds, this non-conserved feature may indicate different architecture of collagens binding in the M. javanica cuticle and, hence, a unique root-knot nematode cuticle structure. (P)

176 Meetings

Protein Expression in Cereal Plants Infested with the Cereal Cyst Nematode, Heterodera avenae

Y. Oka, 1 I. Chet 2 and Y. Spiegel 1 1Dept. of Nematology, ARO, The Volcani Center, Bet Dagan 50250 [Fax: +972-3-9604180]; and

2Dept. of Plant Pathology and Microbiology, The Hebrew University of Jerusalem, Faculty of Agriculture, Rehovot 76000, Israel

Wheat and barley leaf proteins were extracted and then reacted with antisera against pathogenesis-related (PR) proteins. In wheat, infestation by the cereal cyst nematode (CCN), Heterodera avenae, did not result in any detectable difference in protein content of leaves. In barley, however, differences in PR-proteins were recorded in the CCN-infested plants, but these differences could not be related to resistance or susceptibility of the plant. Protein extraction from either barley or wheat roots revealed differences in their profile between CCN-infested and non-infested plants, although it could not be related to resistance/susceptibility of the crop to CCN.

The lectin wheat germ agglutinin (WGA) and barley lectin accumulated in wheat and barley roots infested with CCN, respectively. The lectin WGA was found in the epidermal cells of the root tip and in the nematode feeding site (syncytium) region. Commercial WGA bound to the CCN surface of the nematode. Therefore it is concluded that the lectin plays a role in the nematode-plant interaction.

In the oat root, a protein (45 kDa) recognized by the anti-serum against WGA was induced after CCN invasion to the root. This protein was also induced in the oat root by jasmonic acid exposure, but not by elicitors for PR-proteins or by abscisic acid. The protein level was reduced by inhibition of lipoxygenase activity. Jasmonic acid, therefore, may play a role as a signal for pathogen infestation of the plant. (L)

Involvement of the Nematodes Pratylenchus in Apple and Rotylenchus in Cherry Tree Decline in the Northern Region of Israel

M. Mor, 1 Hadas Cohen 2 and D. Oppenheim 3 1Dept. of Nematology, ARO, The Volcani Center, Bet Dagan 50250 [Fax: +972-3-9604180];

2 MORAG, Golan Heights R&D Authority, Qazrin 12900; and a Extension Service, Ministry of Agriculture, Zefat 13110, Israel

The genus Pratylenchus consists of at least 90 species of plant parasitic nematodes. To date eight species of this genus, including P. penetrans and P..vulnus, have been reported from Israel. A survey carried out in young declining apple groves in the northern region of Israel revealed colonization by large populations of Pratylenchus sp. (90 nematodes/g soil and 3600 nematodes/g roots). Reports from other countries suggested that populations of tens to hundreds of Pratylenchus sp. nematodes per gram of apple roots were damaging to apple trees. The nematodes affect the parenchymatic tissues of apple rootlets, causing the decline of the root system followed by general decline of the trees, small fruits and in severe cases also premature fruit drop. All these symptoms were observed in the damaged groves in which large populations of Pratylenchus sp. nematodes were recorded. Moreover, since no other pathogens were found to be involved in the decline, it is concluded that most probably the large populations of Pratylenchus sp. recorded are involved in apple decline in the survey area.

The genus Rotylenchus consists of at least 80 species of plant parasitic nematodes. To date two species of this genus, R. robustus and R. buxophilus, have been reported from Israel. Nematodes of this genus are ectoparasites of the roots of various plants. Prolonged feeding by these nematodes on


the inner parenchymatic root tissues, damages the roots directly. In addition, the nematodes also cause mechanical damage to the roots, leading eventually to secondary damage by opportunistic pathogens. Surveys carried out during the last year in declining cherry orchards in the Golan Heights revealed colonization by large populations (32 nematodes/g soil) of R. buxophilus. The possible involvement of these nematodes in the decline of the cherry trees is suggested. (L)

Trials to Control Nematodes in a Variety of Crops with Rugby

Y. Benyamini 1 and the Agrochemical Team, 1 D. Orion, 2 Meira Bar-Eyal, 2 Y. Israeli, 3 Esther Hadar 4 and J. Gottlieb 4

1Luxembourg Chemicals and Agriculture Ltd., Tel Aviv 61000 [Fax: +972-3-5100474]; ZDept. of Nematology, ARO, The Volcani Center, Bet Dagan 50250; 3Regional Agricultural Research Center,

Zemah 10985; and 4Extension Service, Ministry of Agriculture, Rehovot 76324, Israel

Rugby (cadusafos) is an OP-nematicide produced by FMC Corp., USA, which was registered in 1995 in Israel for control of nematodes in vegetables and bananas. The product is offered as a 100 g/l microemulsion in water and acts as both a stomach and a contact poison to control a series of nematodes in various crops. Rugby's low water solubility, limited movement in the soil and slow breakdown ensure its long persistence in the root ball. The product is not systemic and therefore there is no hazard of residues in the plant or the fruit, if application is not too close to harvest.

During the last year we have examined the efficacy of the product in the control of nematodes in additional crops by drenching. The present abstract reviews the results obtained in fruit trees, field crops, vegetables and flowers.

In a multiannual trial to control Helicotylenchus multicinctus (the spiral nematode) in bananas, the following products were tested: Rugby, Enzon (CSz), and Nemacur (fenamiphos), injected through the drip irrigation system. Rugby was tested at two dosages, 4 and 6 g a.i./mat. Only the higher rate of Rugby showed a significant and consistent effect on the nematode infection rate of the banana roots. In the second trial season the two Rugby treatments brought about the highest yield gain (17%), of an additional 10 tons/ha. The present year's harvest is not yet complete, but, as in the previous season, the Rugby treatment advanced the onset of flowering significantly.

In experiments to control root-knot nematodes (Meloidogyne spp.) conducted in zucchini, cucumber and tomato, two methods of application were compared: drenching vs spraying and incorporation, at an equal dosage of 30 l/ha. In all the experiments drenching was more advantageous. In the trials with zucchini, the Rugby treatments by both methods of application gave 100% or more increased plant canopy weight compared with control, but as regards the root infestation index, drenching had a clear advantage. In the tomato trial, there was a significant positive influence of the two Rugby treatments on the parameters of infestation, plant canopy weight and fruit yield, but still drenching was superior to spraying and incorporation. Also in the cucumber trial, the best results were obtained by application of Rugby by drenching.

Further trials were conducted to test the efficacy of Rugby to control root-knot nematodes in flower cultures. In trials conducted at Shilat and Moshav haYogev, the state of infestation in roots of Hypericum plants or tomato plants, grown as indicator plants, was recorded. In all the experiments Rugby at 30 l/ha reduced infestation compared with control or the standard Nemacur treatment. Trials and phytotoxicity assays were conducted with additional flower species, such as gerbera, waxflower, kangaroo paw, Asclepias, Auberosa, lisianthus (Eustoma russellianum) and others. In experiments conducted during the last 2 years, it was found that dipping garlic cloves in a 2% Rugby solution caused in many cases accelerated sprouting of the garlic and enhanced weight gain, compared with other treatments. This acceleration obtained when the cloves were sown not too deep, a few days after dipping. (L)

178 Meetings

By title only

The role of the coat protein in the host range of infection by two potyviruses - Eti Meiri, A. Gal'On, A. Gera, V. Gaba and B. Raccah

Alteration of tobacco leaf response to Botrytis cinerea infection by the vesicular arbuscular (VA) mycorrhizal fungus Glomus intraradix- Orna Shaul, S. Galili, Hanne Volpin, Y. Elad, I. Chet and Y. Kapulnik

Trichoderma as a biocontrol agent against root-knot nematodes - Y. Speigel, I. Chet, M. Mor, A. Herela-Esterela, O. Kleifeld, Edna Sharon and Meira Bar-Eyal

Outbreak of fire blight in loquat in Israel - Miriam Zilberstaine, Z. Herzog, D. Zutra, Shulamit Manulis and A. Ein-Gedi

Genomic sequencing of viruses and viroids of plants as a tool in disease diagnosis - A. Ben-Shaul et al.


Abstracts of papers presented at the 8th conference of the Entomological Society of Israel Abstracts of papers presented at the 17th congress of the Israeli Phytopathological Society

Documents