Bacterial Fruit Blotch of Watermelon in Japan And its Seed ...

Bacterial Fruit Blotch of Watermelon in JapanAnd its Seed Health Testing Methods

日本西瓜種子健康檢測

Dr.

National Center for Seeds and Seedlings, Japan

Contaminated seeds with pathogens are very important materials as for the primary source

of the diseases that have an influence of domestic agricultural production and as for the way

that new pathogens come into a country from overseas. In recent years, the situation of the

agriculture of Japan has changed because of enlargement of the scale of plantlet production and

the movement of seed production areas from domestic to other countries, and the international

seed trades have been developed very actively. The pathogen free seeds, therefore, are required

strongly in the international markets. After the bacterial fruit blotch disease (BFB), seed-borne

disease, was observed first at commercial watermelon area in 1998in Japan (Shirakawa et al.,

2000b), the consciousness and attention for the seed-born diseases of vegetables including

cucurbits have been rising more and more in Japan. The BFB disease is one of the most

important diseases that are guarded strictly against entry into Japan from other countries. In this

paper, BFB in Japan and its seed health testing will be introduced.

1.Occurrence

Webb et al. (1965) found first that the causal agent of unidentified disease on watermelon

young plants was seed-transmissible bacteria, and afterward Schaad et al. (1978) reported

that the pathogenic bacteria was Pseudomona preudoalcaligenes subsp. citrulli, new species

of bacteria. Now the name of the bacteria is Acidovarax avenae subsp. citrulli (Aac). In 1987,

BFB was occurred on watermelon fruits in the Mariana Islands, realized as a devastating

disease of watermelon (Wall and Santos, 1988). In the United States, the occurrence of BFB

caused great deal of damage to the watermelon production fields in several states such as

Florida, South Carolina and Indiana State in 1989. BFB can be devastating for the growers

with fruit losses reaching 80 to 100%, and has caused millions of dollars in losses in many

watermelon-producing regions of the United States and other parts of the world. The disease

has spread to different parts of the world, including Brazil, China, Costa Rica, Nicaragua,

Turkey, Thailand, Australia, South Korea, Taiwan, Israel, South Africa and Japan. In

cucurbits, natural occurrences of BFB have been reported on melon, pumpkin and cucumber,

except for watermelon.

Masatoshi SATO

10 日本西瓜種子健康檢測 10-16 (2009)

In Japan, BFB of watermelon was observed first on transplants in transplant houses and

plant leaves in commercial fields at northern area of Honshyu Island, in 1998 (Shirakawa et

al., 2000b). In 2001, 2004 and 2005, BFB was occurred occasionally on watermelon in some

areas. The first occurrences of BFB of melon were observed on leaves or fruits at different

commercial production areas in 2005. The causes of BFB outbreaks in both cases were

inferred the contaminated seed lots produced in other country by the close investigations. In

recent years, BFB disease has never been observed in Japan.

2.BFB symptoms and the causal agent bacteria

A first symptom of BFB in watermelon seedlings is dark water-soaking on lower surface

of cotyledon. These lesions become necrotic, frequently chlorotic halos, resulting in collapse

and death of the seedlings. On watermelon fruit, symptoms appear as small, water-soaked

area, and later enlarge with irregular margins. The lesions turn brown and crack. Leaf lesions

are light brown to dark brown spots with irregular margins, and spread along midrib and main

veins. BFB is very difficult to diagnosis based on leaf symptoms because leaf symptoms may

be often inconspicuous or confused with other common diseases.

Aac is aerobic, gram-negative rods and motile with a single polar flagella. Colonies are

no-fluorescent on King's B medium, and white or cream on most media. The growth occurs at

41 C but not at 42 C.

Walcott et al. (2000b, 2004) reported that there are at least two distinct groups among Aac

by the results of gas chromatografhy-fatty and methyl ester (GC-FRAME) profile and DNA

fingerprinting using pals field gel electrophoresis (PFGE). Kawicha et al. (2002) has

separated four groups based on pathogenicity test by an examination using186 Aac strains.

3.Survival of Aac on watermelon seed

Bacteria on/in infested seeds are significantly important as primary infection of this

disease. Shirakawa et al. (2003) examined the period of Aac survival on the watermelon

seeds at different temperatures of 4 C to 30 C using artificial contaminated watermelon seeds.

Aac could survive at all temperature ranges for more than 26 months, and still kept

pathogenicity for watermelon. For the minimum population of Aac involved in producing

symptoms of watermelon plants, he suggested that there was a possibility that 1 CFU/seed

of Aac made a plant disease when the plant was kept at high humid condition. The population

of Aac is increasing mainly on the leaf surface of seedlings, and it was found that Aac 6 1population in number leached 10 CFU/g of fresh leaf 2 days after inoculation with 10 CFU/

g of fresh leaf.

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4.Methods of diagnosis and seed health testing

For detecting pathogenic bacteria in plants or seeds, several different kinds of techniques

have been developed and employed, as followed. The characteristics of these methods are

shown in Table 1. The method will be selected and used as considering specificity,

sensitivity, purpose, number of samples, equipment and apparatus required and cost.

● -

AacSM (Shirakawa et al., 2000a) is common use for Aac isolation in Japan. The medium

contains are the following per liter: 2.5 g of Na HPO -12 H O, 0.5 g of KH PO , 2.0 g of 2 4 2 2 2 4

(NH ) SO , 10.0g of diammonium adipate, 10 mg of yeast extract, 29 mg of MgSO 7H O, 67 4 2 4 4 2

mg of CaCl , 25 mg of Na MoO -2H O, 12.5 mg of bromothymol blue, 15 g of agar, and 2 2 4 2

antibiotics such as 10 mg of ampicillin, 100 mg of phenethicillin potassium, 2 mg of

novobiocin and 25 mg of cycloheximide. The incomplete AacSM medium is. Incomplete

AacSM, free of both antibiotics of ampicillin and phenethicillin from AacSM medium,

allowes the size of A. avenae subsp. citrulli colony to be bigger than original AacSM without

any influence to the number of present colonies, and results in easier visible distinguishing.

●Serological methods

ELISA kit and immunostrip kit are released by Agdia and/or Adgen for the plant materials.

Latex agglutination test is very simple and useful to determine and identify the Aac colonies

on agar medium.

●Polymerase chain reaction (PCR) PCR is a rapid, highly efficient and sensitive method.

The key of successfully carrying out PCR is a selection of BFB specific primer pairs, and

SEQID4 and SEQID5 (Shaard et al., 2000) and BX-L1 and BX-S-R2 (Bahar et al., 2008) have

been reported.

●Loop-mediated isothermal amplification (LAMP)

LAMP is a new DNA ampl i f ica t ion assay developed by Eiken Chemical

(http://loopamp.eiken.co.jp/lamp/index.html). This assay has unique characters including the

use of 4 different primers and reaction process proceeds at constant temperature (about 65 C)

using strand displacement reaction. The positive reaction of LAMP can be determined

visually by checking the cloud of solution in test tubes.

Semi selective agar medium

12 日本西瓜種子健康檢測 10-16 (2009)

5.Seed health testing methods

The most effective control of BFB is the use of the pathogen free seed lots. In general,

simple, reliable, rapid and affordable seed health testing methods are in demand.

Considering the significant devastation potential of BFB, seed health testing method is

required to be able to enough detect single infested seeds in 10,000 seeds. Sample size of

one seed lot for testing is commonly 10,000 seeds, but, in some cases, has reached 30,000

seeds by demand of seed companies. Seed health testing methods developed for BFB

are described below.

● is widely used today as routine test at seed testing

laboratories. Seeds are grown for 3 weeks under high humid and temperature condition

favorable for symptom development. Seedlings with symptoms are examined by further

analysis for BFB confirmation. This assay has some limitations including 1) time consuming,

2) greenhouse space and labor consuming, 3) overlook of seedlings with weak symptom

expression or without symptoms. And also non-germination seeds are not applicable.

●Sweatbox method is a modified seedling grow-out assay (Koenraadt et al. 2005), in

which seeds are incubated in transplant boxes with high humid and temperature, and

seedling with symptoms are analyzed further for BFB confirmation. Optimum

conditions for rapid development of symptoms can be guaranteed through the use of advanced

climate chambers. This method has advantage to find easily seedlings with symptoms because

healthy seedlings contact easily with infested ones and are infected with Aac in the box. The

problem is that opportunistic microorganisms other than Aac can cause similar symptoms.

Naktuinbouw, Netherlands Inspection Service for Horticulture, employs sweatbox

Seedling grow-out assay

Table 1. Detection and diagnosis methods for Aac and their characters

Methods

Selective agar media

AacSM 2 3 days in Lab○ △

Serological assay

ELISA5

10 3-8 hr in Lab○ △

Immunostrip510 5 min in Lab. & at Field ○ ○

Latex assay710 5 min in Lab. & at Field △ ○

Molecular assay

PCR3

10 4 hr < in Lab◎ △

LAMP3

10 1-2 hr in Lab

(Shirakawa)

◎ △

△：less good, ○：good, ◎：excellent

Specificity Time Cost Applicable situationThresholdCFU/ml)(

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method as a seed health testing method, and issues certificates for BFB.

● is a PCR based method using magnetic

beads combined with antibody, called immunomagnetic beads, from seed washes

(Walcott et al.,2000a,2006). Watermelon seed wash prepared under a vacuum condition

is concentrated with centrifugation, and mixed well with immunomagnetic beads

(IMB). Aac cells are captured with antibody combined with IMB and separated from

seed debris by using magnet. The suspension of IMB can be used as a sample for PCR

after incubation at 100 C. The samples prepared by IMS may be separated with PCR

inhibitors in cucurbit seed extracts leading to the potential for ''false-negative'' reactions.

Bahar et al. (2008) reported that IMS-PCR facilitated the detection of the pathogen

from washings of 5000-seed samples with 0.02% infestation when real-time PCR was

used.

●Membrane filtration immunostaining method,combined a microcolony technique with

alkaline phosphatase conjugated antibody visualization system, was applied for the Aac

detection in watermelon seeds by Matsuura et al.(2008). Seed washing treated with an

ultrasonic cleaner is filtered though a 0.45 mm cellulose nitrate filter. The membrane is placed

onto AacSM selective medium and incubated for 3 days. The membrane is stamped on a new

AacSM medium, and overlaid with conjugated antibody resulting in deep purple reaction

(NBCP/NBT system) that enables detection. Living Aac can be obtained from the membrane

on the new AacSM medium before immunostaining. It is reported that the detection

threshold of this method for Aac was several CFU/100ml using 1,000 commercial watermelon

seeds.

● Membrane filtration - LAMP assay for the Aac detection in watermelon seeds was

developed by Oya et al. (2008). Seed wash treated with an ultrasonic cleaner is filtered

though a 5.0 mm filter. After the filter is washed well with a vortex mixer, the wash is

concentrated with centrifugation and used for LAMP assay. The detection threshold of 3

this assay is approximately 10 CFU/ml. The authors confirmed the presence of the

BFB pathogen in imported commercial watermelon seed lots by this assay. This assay needs

only about 2 hr to carry out completely and is thought to be applicable to seed

inspection at plant quarantine.

There are some points to achieve reliably seed health testing for BFB by using DNA

amplification-based method. The first is to isolate the pathogenic bacteria from the

samples that indicate positive reaction. It is difficult to tell whether seed samples

showing positive are contaminated or not, because this assay can detect the dead

bacteria because of DNA amplification. DNA amplification-based assay can' t

distinguish between dead and live cells. The best way to confirm the results is that

isolation of living cells on agar medium is involved in the testing. The second is to detect

the pathogens surviving at internal seeds. Although the location of Aac on/in the seed

have not yet found in detail, it is thought that Aac may survive at internal seeds as

reported by Rane and Latin(1992). Some kinds of methods such as seed processing or

Immunomagnetic separation-PCR (IMS-PCR)

14 日本西瓜種子健康檢測 10-16 (2009)

seed treatment to detect the pathogen at internal seeds need to be taken instead of

preparation of seed washes.

We have been developing a new method for BFB seed health testing, called sweat-bag

seedling method(Sato et al, 2006). This method consists of enrichment phase and

detection phase using both of PCR and semi-selective agar medium. I believe that this new

method will be able to help to solve the problems mentioned above. The progress of

this method is ongoing.

6.Conclusions

Since BFB is still the most important disease that many companies in the world pay

a lot of attentions, the significance of the seed health test has been fully recognized. Seed

companies in Japan are utilizing the certificates for commercial seed lots, as the way

of seed quality management, because of reducing their risks. For improvement of the

reliability of the test for the cucurbits, it is necessary to obtain some information, as

like watermelon, including (i) distribution and location of the pathogen at internal

seed,(ii)population dynamics of the pathogen on seeds and seedlings and (iii) minimum

population of the pathogen causing disease. We will need to find these relationships lying

between the pathogenic bacteria and seeds in future.

Literature cited

Bahar, O.,Efrat,M.,Hadar, E., Dutta,B., Walcott,R.R. and Burdman, S.(2008)

New subspecies-specific polymerase chain reaction-based assay for the detection of

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Koenraadt, H, Borst, R., van Vliet, A., Hoekstra,M.,van Schie and J., Buimer,M.(2005)

Detection of Acidovorax avenae subsp. citrulli with the sweatbox method. Phytopathology

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Matsuura, T., Shirakawa, T., Sato, M., Inoue, Y. and Azegami, K. (2008) Detection and

isolation of Acidovorax avenae subsp. citrulli from watermelon seeds using membrane

filtration immunostaining. Jpn. J. Phytopatol. 74: 1153-156.

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