Benefits of in vitro “biotization” of plant tissue cultures with microbial inoculants

In Vitro Cell. Dev. Biol.--Plant 34:122-130, April-June 1998 �9 1998 Society for In Vitro Biology 1071-2690/98 $05.00+0.00

REVIEW BENEFITS OF IN VITRO "BIOTIZATION" OF PLANT TISSUE CULTURES WITH

MICROBIAL INOCULANTS

JERZY NOWAK

Department of Plant Science, Nova Scotia Agricultural College, P.O. Box 550, Truro, Nova Scotia, Canada B2N 5E3

(Received 8 August 1997; accepted 18 December 1997; editor T. A. Thorpe)

SUMMARY

In vitro cocuhure of plant tissue explants with beneficial microorganisms induces developmental and metabolic changes in the derived plantlets which enhance their tolerance to abiotic and biotic stresses. The induced resistance response caused by the inoculants is referred to as "biotization." There is enough experimental evidence with bacteria (bacterization) and vesicular arbuscular mycorrhiza (mycorrhization) inoculations to recommend utilization of this technology in commercial micropropagation. The paper reviews literature on this topic, outlines other uses and potential uses of in vitro plant- microbial cocuhures, and discusses their current constraints.

Key words: micropropagation; bacterization; mycorrhization; stress resistance responses.

INTRODUCTION

Plant propagation technology via tissue culture has been developed over the last 30---40 yr as a spinoff of in vitro studies on differ- entiation (Pierik, 1987, 1988). Typically, aseptic explants are grown under low light intensity in small containers, on artificial culture media containing sucrose, mineral salts, vitamins, and growth regu- lators, in concentrations exceeding levels recorded under natural environments (Pierik, 1987; Leifert et al., 1995). Moreover, the culture conditions do not allow for a good gas and moisture exchange (McClelland and Smith, 1990; Jeong et al., 1995; Matthijs et al., 1995). This causes developmental distortions (Ziv, 1991; Van Huy- lenbroeck and Debergh, 1996) and repression or modulation of some metabolic pathways (Phan and Hegedus, 1986; Baccou, 1995; Des- jardins, 1995; Rchid and Baccou, 1995). Plantlets have reduced photosynthetic capacity (Cournac et al., 1991, 1992; Desjardins, 1995), malfunctioning stomata (Ziv et al., 1987; Frommel et al., 1991a; Majada et al., 1995), root system devoid of root hairs, poor cuticle development, and low wax deposits (Pierik, 1987; Lee and Wetzstein, 1988; Preece and Sutter, 1991). Consequently, when transplanted out of the culture vessels, they suffer from severe environmental stresses and substantial losses may occur (Preece and Sutter, 1991; Van Huylenbroeck and Debergh, 1996). Manipulation of acclimation conditions prior to or upon transplanting usually reduced losses (Donnelly et al., 1985; Desjardins et al., 1988; Preece and Sutter, 1991; Desjardins, 1995; Van Huylenbroeck and Debergh, 1996), however, at additional cost to the producer. To minimize these constraints, in recent years, attempts have been made to develop photoautotrophic culture systems with high light intensity and reduced or eliminated sugar in the medium (Kozai et al., 1988; Kozai, 1991; Fujiwara and Kozai, 1995; Jeong et al., 1995). So far, this technology has not gained popularity.

During the last decade some researchers have reached for natural inhabitants of plants, epiphytes and endophytes, to enhance adap-

tation of tissue culture propagules to environmental stresses (reported by Herman, 1996a, 1996b). The plant-associated microorganisms, endophytes in particular, were always treated as problem-causing contaminants of in vitro cultures (reviewed by Cassells, 1991, and Leifert et al., 1994) and various procedures have been developed for their elimination (Cassells, 1991; Reed and Tanprasert, 1995) or management (Levin et al., 1997).

In nature, microorganisms inhabit the interior and exterior of plant organs (Jacobs et al., 1985; Frommel et al., 1993; Leifert et al., 1994; Mclnroy and Kloepper, 1995; Sturz, 1995; Sturz and Christie, 1995; Sturz et al., 1997). Some of these microorganisms, plant-beneficial bacteria and vesicular-arbuscular mycorrhizae in particular, can im- prove plant performance under stress environments and consequently, enhance yields (Brown, 1974; Schippers et al., 1987; Kloep- per et al., 1988, 1989; Lynch, 1990; Frommel et al., 1991b; Pfleger and Linderman, 1994; Chen et al., 1995; Datnoff et al., 1995; Glick, 1995; Balachandran et al., 1997; Bethlenfalvay et al., 1997; Creus et al., 1997; Jackson et al., 1997; Lazarovits and Nowak, 1997; Zehn- der et al., 1997). The mechanisms governing these responses have been extensively reviewed (e.g., Kloepper, 1988, 1989; Lynch, 1990; Dowling and O'Gara, 1994; Pfleger and Linderman, 1994; Glick, 1995, 1997; Lazarovits and Nowak, 1997). This review provides an outline of the present status of knowledge on utilization of microbial inoculants in plant tissue culture.

IN VITRO "BIOTIZATION"

Induction of stress resistance in plant propagules produced in vitro prior to transplanting, is a primary target of several researeh groups attempting utilization of microbial inoeulants in micropropagation (Hooker et al., 1994; Elmeskaoui et al., 1995; Nowak et al., 1995; Balla et al., 1997; Lazarovits and Nowak, 1997; Murphy et al., 1997; Wilhelm et al., 1997). Induced resistance responses to microbial inoeulants are well documented in plants grown in vivo (reviewed by Benhamou, 1996; Benhamou et al., 1996; Hunt and Ryals, 1996; Kloepper et al., 1997). Such responses in vitro have only been ree-

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BIOTIZATION IN TISSUE CULTURE 123

ognized very recently (Herman, 1996b) and are referred to as "biotization" (reported by Herman, 1996a). I propose to define this term as follows: Biotization is a metabolic response of in vitro-grown plant material to a microbial inoculant(s), leading to the developmental and physiological changes enhancing biotic and abiotic stress resistance of the derived propagules. Establishment of artificial symbiotic associations (see Varga et al., 1994, and Preininger et al., 1997) could also belong to this category.

Bacterization. Eleven yr ago we initiated in vitro bacterization of potato plantlets to enhance their transplant stress tolerance and elim- inate an expensive greenhouse hardening step, which even now is commonly used by pre-elite seed potato producers (first reported by Herman in 1987). A pseudomonad bacterium, originally isolated as a contaminant from surface-sterilized onion roots in a study of Glo- mus vesiculiferum effect on in vitro growth of potato plantlets (Nowak et al., 1997), appears to be the most effective plant growth promoter we have come across so far (Conn et al., 1997; J. Nowak and C. Smith, unpublished). The bacterium (Pseudomonas spp. strain PsJN) does not grow on a standard plant tissue culture medium without the presence of the plant, and forms endophytic and epiphytic populations when cocultured, either with potato (Frommel et al., 1991a) or tomato (Pillay and Nowak, 1997). Thus, in clonally propagated material there is no need for further reinoculation (Frommel et al., 1991a; Nowak et al., 1997). The bacterium stimulates plantlet growth (Frommel et al., 1991a; Liu et al., 1995; Nowak et al., 1995; Pillay and Nowak, 1997; Bensalim et al., 1998) and induces developmental changes (Frommel et al., 1991a), leading to better water management (Nowak et al., 1995; Lazarovits and Nowak, 1997) and enhanced resistance to low levels of pathogens (Nowak et al., 1995; Richards, 1997; Stewart, 1997; Sharma and Nowak, 1998) upon transplanting. Typical modifications are presented in Figs. 1 and 2. The inoculated plantlets have a massive, well-branched root system (Fig. 1 A), with bacteria cells grouped in small clusters along the older roots (top portion of Fig. 1 B) and forming tight cover around newly emerging root branches (bottom part of Fig. 1 B). After 4 wk in culture, compared to the nonbacterized controls, the bacterized plantlets are de- velopmentally more advanced, have sturdier stems, with more lignin deposits around the vascular system (Fig. 2 A vs B), more root hairs (Fig. 2 C vs D) and more and larger leaf hairs (Fig. 2 E vs F). Stomata function of the bacterized in vitro plantlets closely resembles that of greenhouse-hardened transplants. Five rain after leaf detachment from in vitro-grown plantlets, 90.1% of the stomata were closed in the bacterized versus 8.3% in the nonbacterized controls; the figures for the greenhouse-hardened transplants were 89.3 and 88.7% for the bacterized and controls, respectively (Frommel et al., 1991a). Improvement of water relations under osmotic stress was also recorded in wheat seedlings cocuhured with AzospiriUum brasiliense strain SP245 (Creus et al., 1997). Live bacterial cells were required to induce these responses. Neither growth promotion nor developmental changes were induced, either by the autoclaved bacteria (Frommel et al., 1991a; Creus et al., 1997) or by the culture medium (Frommel et al., 1991a). Moreover, the growth stimulation pattern in tomato was closely related to the endophytic bacterial colonization (Pillay and Nowak, 1997). There was no relationship between colonization of the root surface and growth promotion in plantlets of tomato (Pillay and Nowak, 1997), potato (J. Nowak, S. K. Asiedu, and C. Smith, unpublished), or other vegetable crops (Liu et al., 1995).

In 8 of 11 experiments conducted with potato plantlets transplanted directly from culture vessels to the field between 1987 and

1992, bacterized plantlets had significantly better survival than nonbacterized controls (Nowak et al., 1995). Improved survival has also been recorded by Balla et al. (1997) with rooted black locust (Robinia pseudoacacia L.) plantlets inoculated with different Rhizobium isolates selected for growth vigor from a population of root nodule bacteria obtained from five Robinia varieties. The bacterized potato transplants had also higher vigor during the first few weeks after planting to the field (e.g., Fig. 1 C). However, tuber yield responses in the field varied from yr to yr. In most experiments, there was an earlier tuber set and an enhancement of tuber number and yield in the bacterized treatments when compared to the nonbacterized (J. Nowak, S. K. Asiedu, and coworkers, unpublished). High precipi- tation or a very severe drought usually reduced this effect. Green- house experiments also demonstrated that plants derived from dual cultures of potato and the pseudomonad bacterium had a larger root system, set stolons and tuberized earlier, and gave better tuber yield than nonbacterized controls (Dunbar, 1997). Both in vitro and ex vitro benefits of bacterization depended on plant species, cultivar, and growth conditions (Nowak et al., 1995, 1997; Dunbar, 1997; Laza- rovits and Nowak, 1997; Pillay and Nowak, 1997; Bensalim et al., 1998).

Agrobacterium rhizogenes stimulation of adventitious rooting in culture is a well-known phenomenon (see McAfee et al., 1993; Bums and Schwarz, 1996). Rooting stimulation by bacteria isolates consid- ered to be culture contaminants (other than A. rhizogenes) have been reported only recently. In our group, dramatic root growth promotion was induced by a pseudomonad bacterium cocuhured with herbaceous plants, potato (Frommel et al., 1991a; Lazarovits and Nowak, 1997), tomato (Pillay and Nowak, 1977), watermelon (Liu et al., 1995), and cucumber and pepper (Nowak et al., 1995). Bums and Schwarz (1996) had good success with induction of root development on slash pine seedling explants. The authors were able to root 85, 50, and 15% of explants of 6, 16, and 21 wk of age, respectively, when cocultured with their bacterium isolate. None of the explants rooted in the nonbacterized control groups. Similar to our observations with herbaceous plants, Burns and Schwarz (1996) reported that the bacterium-induced roots resemble "seedling" roots rather than "hairy" roots, which are known to be induced by A. rhizogenes. Contrary to our observations that cocuhured plantlets had always well-branched, massive, but usually shorter root systems than noninoculated controls, Lifshitz et al. (1987) reported dramatic elonga- tion of roots in Brassica campestris seedlings by a strain of Pseudomonas putida.

In vitro cocultivation of soybean cotyledon explants with two strains of Pseudomonas maltophilia, stimulated development of nodular callus with high regeneration potential (Yang et al., 1991). Im- provement of somatic embryogenesis by selected bacterial inoculants in genotypes recalcitrant to regeneration has further been reinforced by a study by Visser-Tenyenhuis et al. (1994). In a study by Shetty et al. (1995), shoot explant bacterization of oregano with a Pseudo- monas spp. prevented vitrification. The oregano plantlets cocuhured with the bacterium had lowered water content and contained more phenolics and chlorophyll than nonbacterized controls. Similar responses were recorded in our studies with potato (Nowak et al., 1997) and vegetable crops (Nowak et al., 1995; Pillay and Nowak, 1997). Bacterized plantlets were greener, had elevated levels of cytokinins (Lazarovits and Nowak, 1997), phenylalanine ammonia lyase (PAL), and free phenolics (chlorogenic and caffeic acids in particular) (No- wak et al., 1997; Richards, 1997) and contained more lignin (From-

124 NOWAK

A B

CONTROL BACTERIZED

C

BACTERIZED CONTROL

FIG. 1. In vitro (A) and ex vitro (C) responses of potato plantlets, cv. Kennebec, to explant bacterization with a Pseudomonas sp., strain PsJN, and the bacterial colonization of roots in vitro (B). A, In vitro responses; explants taken from 6-wk-old stock plants were inoculated with approximately 3 • 10 a cfu mL -1 bacteria in phosphate-buffered saline (PBS) or treated with PBS only (control) as in Frommel et al. (1991a) and grown either on sucrose (S) or lactose (L) containing MS medium for 4 wk. B, Bacterial colonization of older (top) and younger (bottom) branching roots in a 4-wk-old culture as above on sucrose as a carbon source. Root sections were fixed in 4% glutaral- dehyde in 0.07 M phosphate buffer, pH 6.8, overnight, washed with the buffer, dehydrated with acetone, dried to a critical point, sputter coated with 15 Ilm gold/palladium and viewed with a ]EOL scanning electron microscope (SEM) under 3500 • magnification (bar = 5 p.m). C, Ex vitro bacterized and control tissue culture plantlets planted directly from Magenta culture vessels to the field 5 wk after transplanting. The experiment was conducted on Banting Field at the Nova Scotia Agricultural College, Trurn, NS, Canada, in 1990.


CONTROL BACTERIZED

FIG. 2. Bacterization induced developmental changes in potato plantlets: A and B, Lignin deposits (arrows) around vascular system in 4-wk-old nonbacterized (A) and bacterized (B) Kennebec plantlets. Stem sections taken 5 mm above the node of the original nodal explant were fixed in formalin-aceto-alcohol; tissue was dehydrated with ethanol, anhydrous ethanol/xylene, and xylene at 4 ~ C and cast in paraffin. Sections 10-/.tin thick were trimmed with a rotary microtome, placed on glass slides in a Haupt's A solution, flooded with Haupt's B solution, placed on a slide warmer (45 ~ C), dried for 24 h, stained in 0.1% toluidene blue as in O'Brien and McCully (1981), and viewed with a light microscope under 100 X magnification (bar = 200 p_m). C and D, SEM magnification of primary roots of 4-wk- old Kennebec cultures; 150 • magnification (bar = 100 ~tm). E and F, SEM magnification of leaf surface of the cultures above; 50 X magnification (bar = 500 p_m).

126 NOWAK

mel et al., 1991a; Richards, 1997). Cocuhuring chestnut with a strain L25 of Bacillus subtilis, Wilhelm et al. (1997) also found production of pathogenesis-related proteins, extracellular chitinase, and 13-1,3 gluconase isoenzymes which did not appear in wounded control plants. The above are typical symptoms of induced resistance responses (Dixon et al., 1994; Hunt and Ryals, 1996). In our study with potatoes, elevated PAL activity in bacterized plants, compared to nonbacterized, was found during the first 4 wk of growth; there was no difference in its level in 6-wk-old plantlets. However, when explants were taken from 6-wk-old bacterized and control stock plants, PAL activity in the newly derived shoots was higher in the prebacterized treatment (Nowak et al., 1997; Richards, 1997). This indicates a more rapid plant response to cutting in the prebacterized tissues. The speed and degree of plant response to stress signals is recognized as a critical factor in plant resistance to pathogen attack (Behamou, 1996). Unlike the Bacillus strain in the study by Wilhelm et al. (1997), our Pseudomonas bacterium did not show any antagonistic effects on fungal pathogens in in vitro plate bioassay. Moreover, bacterized potato were resistant to low levels of Verticillium albo- atrum (Nowak et al., 1995; Stewart, 1997) and tomato plants to V. dahliae (Sharma and Nowak, 1998). Plantlet bacterization, upon transplanting, did not induce such responses (Stewart, 1997; Sharma and Nowak, 1998). As the in vitro growth responses depended on the degree of endophytic colonization and a certain threshold of the bacteria concentration was required to trigger such response (Pillay and Nowak, 1997), Sharma and Nowak (1998) postulated similar induction patterns for the resistance to pathogens. There is some evidence that a pathway of responses to biotic and abiotic stresses in plants can be universal (reviewed by Sheen, 1996, and Harmon, 1997).

Mycorrhization. Benefits of inoculation of tissue culture plantlets with endomycorrhizal fungi have been advocated by several authors (Pierik, 1988; Ch~vez and Ferrera-Cerrato, 1990; Ponton et al., 1990; Hooker et al., 1994; Elmeskaoui et al., 1995). It is well established that root colonization by vesicular-arbuscular mycorrhizae (VAM) im- proves plant nutritional status, disease resistance, and water management (see references in Powell and Bagyaraj, 1984; Hooker et al., 1994; Elmeskaoui et al., 1995). In an attempt to harness this potential for the improvement of the performance of tissue culture plantlets, several researchers inoculated rooted shoots upon transplanting (Ch~vez and Ferrera-Cerrata, 1990; Ponton et al., 1990; Wang et al., 1993 and references therein). The benefits of mycorrhization depended on the growing medium (Ponton et al., 1990) and the degree of root colonization (Wang et al., 1993). As the VAM stimulation of plantlet growth was frequently expressed only after acclimatization, Wang et al. (1993) suggested development of a culture system into which the mycorrhizal fungi are introduced in vitro, during the rooting stage. Challenges of the axenic cultivation of VAM fungi were overcome in studies by B~card and Fortin (1988) and B6card and Pich~ (1989). The authors were able to successfully grow the fungi in vitro on Ri T-DNA transformed carrot roots, under elevated CO2. Capitalizing on this technique, Y. Desjardins's group developed a "tripartite" culture system for in vitro inoculation of strawberries (Desjardins, 1995; Elmeskaoui et al., 1995) and vegetable crops (Y. Desjardins, personal communication).

The most effective treatment consisted of a 30-d-old VA (Glomus intraradices) mycorrhizal-transformed carrot root culture and strawberry shoots in cellulose plugs. After root induction, the plugs were placed on the surface of the mycorrhized root culture and grown in a growth chamber, under 5000 ppm CO2 for 20 d (Elmeskaoui et al.,

1995). All plantlets were successfully colonized and exhibited larger root systems, better shoot growth, and higher (more negative) osmotic potential than nonmycorrhizal controls. The authors suggest that the enhancement of osmotic potential can be an important preadaptation step prior to full acclimation of plantlets for transplanting. Indeed, the work by Creus et al. (1997) demonstrated that a higher potential recorded in wheat seedlings cocultured with Azospirillum allowed them to withstand osmotic stress much better than noninoculated controls with lower osmotic potential.

Another mycorrhization system was developed by A. Cassell's group (Murphy et al., 1997). The authors grew strawberry microplants in a polyurethane foam substrate under photoautotrophic conditions, with reduced sucrose concentration in the medium. The foam tear- away strips seem to be very useful as they can be made to fit any culture vessel and the VAM spores can be placed directly in the planting holes. The mycorrhized plantlets had better establishment and produced more runners than nonmycorrhized controls. Murphy et al. (1997) also suggest that a combination of VAM and beneficial bacteria can perhaps be even more efficient than VAM alone.

Recent reports by Walley and Germida (1997) indicate that utilization of such combinations should be approached with caution. In their study with five plant growth-promoting pseudomonads and a VAM isolate NT4 of Glomus clarum in a gnotobiotic system, dual inoculation with VAM and bacteria usually reduced the magnitude of the plant growth response as compared to the single inoculant. Moreover, two bacteria strains inhibited G. clarum NT4 spore germination.

Other potential inoculations. Cyanobacterial and fungal culture extracts have been proven to induce developmental changes and pathogen resistance in tissue-cultured material when added to the medium. Both dialysates and nondialysates of hot water extracts from filamentous cyanobacteria, Nostoc sp. and Anabaena sp., and unicellular strains of Synechococcus sp. and Xenococcus sp., promoted somatic embryogenesis and plantlet regeneration in Daucus carota L. (Wake et al., 1991). Higher molecular compounds (nondialysates) from the unicellular strains were more effective than the smaller ones (dialysates). Further fractionation of the extract from Synechococcus sp. strain NKBG 042902 by centrifugation allowed the authors to concentrate this high molecular elicitor (Wake et al., 1992). When added to an embryogenic culture of carrot it induced chlorophyll accumulation (5 X more than in nontreated cultures) and adjusted the chlorophyll a/b ratio close to the zygotic embryo level. Plantlet conversion was 60% with this compound and only 28% without its addition.

Yu et al. (1990) demonstrated the stabilizing effect of Verticillium albo-atrum culture filtrate on somatic embryogenesis in alfalfa cell suspension culture. While control cultures lost their somatic embryogenesis after six subcultures, the culture filtrate-treated ones (20% vol/vol) still maintained a relatively high number of embryos through all subcultures. Shoot regeneration from potato tuber discs was also stimulated by a culture filtrate of Alternaria solani (Lynch et al., 1991). The authors advocate it for use in in vitro selection and genetic transformation of this species.

Successful application of microbial culture filtrates in control of plant tissue culture contaminants was demonstrated by Hussain et al. (1994). The acetone-precipitated fraction of Bacillus subtilis Ehrnberg (strain 2) and Trichoderma viride Pers. (strain A) exhibited high antifungal activity, whereas extracts from Pseudomonasfluores- ceace Migula (strain X) had high antibacterial activity. The bacterial


extracts did not reduce the performance of uncontaminated cultures of Nicotiana tabacum L. over four subcultures and reduced the growth of accidental contaminants. Heat-stable mycelial extracts of another Trichoderma species, a nonpathogenic T. longibrachiatum, were capable of triggering induced resistance responses in tobacco seedlings to Phytophthora parasitica var. nicotianae (race 0) (Chang et al., 1997). It induced expression of pathogenesis-related proteins, PR-16 and osmotin (PR-5) on a higher level than an extract prepared the same way from the pathogenic fungus. Also the elicitation of the defense responses could not be fully achieved by a combination of salicylic acid and methyl jasmonate (Chang et al., 1997).

The above studies further emphasize a potential for use of nonpathogenic organisms in plant tissue culture systems. If the culture conditions are refined, artificial associations with either individual microorganisms or their groups can be created. Such microorganisms not only would act as inducers of the stress resistance responses but also could occupy microsites on host plants, making them unavail- able to pathogens. An example of a successful artificial association between plants and a nitrogen-fixing bacterium has been described by Varga et al. (1994). The authors created a symbiotic culture system, callus-bacterium, between Daucns carota L. and Azotobacter zettuovii (CRS-H6) which could grow for 4 yr on a nitrogen-free medium with lactose as a carbon source. The bacterium, located in intracellular spaces, could also be transmitted to and fix nitrogen in newly regenerated plantlets (Varga et al., 1994; Preininger et al., 1997). Recently the authors were able to establish similar associations also in tomato, potato, wheat, sugar cane, and poplar, using 11 strains of 8 Azotobacter species (Sz. S. Varga, personal communication) and in strawberries with Azomonas insignis (Preininger et al., 1997). In the tomato system, the bacterium could also be transmitted through seeds; approximately 20% of seedlings derived from the seeds of Azotobacter beijerinckii containing tomato demonstrated N 2

fixing activity 2 mo. after germination (Sz. S. Varga, personal communication).

A potential also exists for the utilization of traditional "night- mares" of micropropagators, the mycoplasma-like organisms, in recent literature termed as 'mollicutes' (Bove and Gamier, 1997), and viruses (Herman, 1996b). Both are known to form very stable associations with plants and are difficult to eradicate in tissue culture. Reports on mycoplasma promotion of morphogenesis in sandalwood (Santalum album) callus, and tomato mosaic virus stimulation of callus growth and morphogenesis in five Lycopersicon species (see Herman, 1996b) as well as viral modification of plant responses to environmental factors (Balachandran et al., 1997) encourage further investigation of their potential use in tissue culture.

PLANT--MICROBIAL COCULTURES FOR PURPOSES

OTHER THAN PROPAGATION

In vitro bioassays. Stimulation of Brassica campestris root development in a gnotobiotic growth pouch bioassay by pseudomonad bacteria (Lifshitz et al., 1987) indicated the usefulness of such a system in screening of rhizosphere bacteria for plant growth promotion without interference of contaminants. DeFreitas and Germida (1990) further demonstrated modification of root morphology and stimulation of root hair development in root tissue culture of wheat by various bacterial species and strains, and Chanway and Nelson (1991) showed soybean callus growth promotion on a vitamin-free medium by one of the two tested strains of Pseudomonas putida. Recently, a

canola and lettuce seeding bioassay was also used to demonstrate root development stimulation of nonlegume plants by Rhizobium leguminosarum (Noel et al., 1996). One of the disadvantages of seed- based bioassay is the difficulty with obtaining "clean" cultures. Conn et al. (1997) described a clonal bioassay based on bacterization of potato nodal explants. Such bioassay, modified after Frommel et al. (1991a), provides genetically identical explants which are taken from contaminant-free stock plants of the same developmental stage, is reproducible and allows study of bacteria-plant interactions for many wk. The authors (Conn et al., 1997) were able to successfully assess growth-promoting ability of various bacterial isolates and Tn5 mu- tants of a nonfluorescent Pseudomonas species (PsJN). The bioassay can also be used to determine plant genotypic responses to particular strains of bacteria or their combinations.

Plant culture bioassays are also recommended for identification of bacteria for biological control organisms (e.g., a surface-sterilized petunia disc bioassay for Botrytis-controlling bacteria reported by Gould et al., 1996) and for monitoring disease development on roots (e.g., Verticillium development on roots of tissue culture-grown potatoes reported by Platt and Bollen, 1993). Hampson et al. (1997) were recently able to establish a successful dual culture system of potato and the infective agent of potato wart disease (Synchytrium endobioticum) which can be used for virulence determination and identification of phenotypes of this pathogen.

Test-tube ecophysiology of plant-microbial interactions. Erratic yield responses of field-grown crops subjected to microbial inoculants frustrate the inoculants' users and producers (Rovira, 1991; Lazarovits and Nowak, 1997). One of the limitations preventing us from using their full potential is insufficient knowledge of the dy- namics of microbial interactions in plant rhizosphere and phyllos- phere under varied environmental conditions. Tissue culture allows for the creation of simple model systems for testing single factors and their combinations. A recent study by Pillay and Nowak (1997) demonstrated, for example, that lower inoculum densities were required to maximize root growth promotion (approximately los cfu mL -~) than shoot growth promotion (approximately 3 • l0 s cfu mL-1) in tomatoes cocuhured with a growth-promoting pseudomonas bacterium. Temperature had also a dramatic effect on the degree of plant growth promotion (Pillay and Nowak, 1997; Bensalim et al., 1998) and tissue colonization (Pillay and Nowak, 1997). Compared to room temperature, a temperature elevation stimulated in vitro growth responses to bacterization in most clones of potato (Bensalim et al., 1998) but lowered them in tomatoes (Pillay and Nowak, 1997). In potato, under heat stress, the bacterium facilitated changes in biomass allocation to roots (Bensalim et al., 1998). In both these studies, there were dramatic differences between the responses of different cultivars. This could be partly linked to the genotypic differences in ethylene accumulation in culture vessels (J. Nowak and D. Stevens, unpublished).

Carbon source in the medium can also have an effect on plant growth responses to bacterization. Pseudomonas sp. strain PsJN, as well as other growth-promoting rhizobacteria tested in our laboratory, including some strains of Rhizobium leguminosarum (J. Nowak, T. Sturz and D. Stevens, unpublished), always stimulated more root growth on sucrose and more shoot growth on lactose-containing medium (Fig. 1 A).

A beneficial effect of bacterial combinations on in vitro growth of red clover was demonstrated by Sturz et al. (1997). The authors identified root nodule bacteria (isolated from in vivo-grown plants) be- longing to the genera Agrobacterium, Bacillus, Pseudomonas and

128 NOWAK

Curtobacterium as capable of in vitro seedling growth promotion or nodulation when inoculated either singly (growth promotion) or in combination with Rhizobium leguminosarum BV trifolii (growth promotion and/or nodulation). Interestingly, the Rhizobium inoculation alone always caused seedling growth depression compared to noninoculated controls.

The above-outlined studies are examples of the potential uses of the tissue culture systems for study of allelopathic side effects of different microbial combinations on plant performance, selection of the best strain combinations, determination of inoculum concentrations, and plant growth conditions for bacteria utilization (endophytes in particular, as their populations are less susceptible to environmental changes) to their full potential. Similar studies are also being conducted with VAM (Desjardins, 1995) and VAM-rhizobacteria combinations in other laboratories (Y. Desjardins, personal communication; Walley and Germida, 1997).

CONSTRAINTS AND FUTURE DIRECTIONS

The fact that we can culture probably only a few percent or even less of naturally occurring microorganisms (Hugenhohz and Pace, 1996) is one of the challenges of the utilization of microbial inoculants in plant production. Development of new culture methods al- lowing establishment of stable associations between plants and beneficial organisms in Vitro and ex vitro and understanding of mechanisms of signal recognition and transduction in plant-microbial associations under different environments are probably the most critical elements of this challenge. There are also psychological and regulatory barriers set up, for example, in the nuclear seed potato systems which do not allow cocuhuring of tissue culture propagules with microorganisms. In view of the present knowledge of the role of these microorganisms and a substantial body of literature demon- strating either their benefits (reviewed above) or passivity (e.g., Mo- nette, 1986), the regulatory hurdles should be lifted. Such a move would stimulate inflow of industrial research funds. This in turn could stimulate research aiming at the genetic modification of the plant-associative microorganisms, as well as modification of plant genome to target specific groups of such microorganisms. Plant breeding for sustainable agricultural production systems must seri- ously consider such an option. Certain groups of microorganisms could not only benefit a targeted crop but also other crops in a rotation system. In a study with potato, clover, and barley, we have identified some endophytic bacteria, including Rhizobia, which can benefit all three crops (J. Nowak, A. V. Sturz, and B. R. Christie, in preparation). A field study by Bethenfalvay et al. (1997) demonstrate that mycorrhizal fungi-rhizobacteria combinations not only can affect plants directly but also indirectly, by the enhancement of the proportion of water-stable aggregates in soils.

ACKNOWLEDGMENTS

Microscopy work presented in Figs. 1 and 2 was done by Mr. Jeff Richards, a graduate student (M.Sc.) working under the author's cosupervision. The author would like to thank Ms. Marie Marshall for her contribution in pre- paring the manuscript.

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