Microbial Inoculants as Crop-Yield Enhancers

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  • Volume 6, Issue 1 (1987) 61

    MICROBIAL INOCULANTS AS CROP-YIELD ENHANCERS

    Authors: Yaacov Okon Yitzhak Hadar Department of Plant Pathology and Microbiology

    Faculty of Agriculture The Hebrew University of Jerusalem Rehovot, Israel

    Referee: Ralph Baker Department of Plant Pathology and Weed Science Colorado State University Fort Collins. Colorado

    I. INTRODUCTION

    Various soil microorganisms that are capable of exerting beneficial effects on plants or antagonistic effects on plant pests and diseases either in culture or in a protected environment have a potential for use in agriculture and can lead to increased yields of a wide variety of crops. However, this ability does not always ensure that the release of the organisms into an environment, such as soil, will produce the desired results.

    This review describes some groups of beneficial microorganisms which are currently used in commercial agriculture or which may become practical for use in the future. Yield increases are brought about by these microorganisms by several modes of action. Microbial groups that affect plants by supplying combined nitrogen (N) include the symbiotic N-fixing rhizobia in legumes, actinomycetes in nonleguminous trees, and blue-green algae in symbiosis with water ferns. In addition to supplying combined N by biological N,-fixation, free-living N-fixing bacteria of the genus Azospirillum affect the development and function of grass and legume roots, thus improving mineral (NO;, P O , and K+) and water uptake. Other microorganisms that are known to be beneficial to plants are the phosphate solubilizers, plant-growth-promoting pseudo- monads, and mycorrhizal fungi.

    Indirect effects on crop yield can be obtained by inoculation with microorganisms capable of reducing damage caused by pathogens and pests. These groups include bio- control agents of soil-borne pathogens such as Agrobacterium radiobacter and Tri- choderma, bacterial and fungal insecticides, nematode-trapping fungi, microbial her- bicides, and microbes that compete with ice-nucleating bacteria, thereby preventing frost damage to leaves. In this review, the subject of the application of chemicals of microbial origin such as antibiotics or the toxin produced by Bacillus thuringiensis is not included.

    The use of these microorganisms is of economic importance to modern agriculture as they can replace costly mineral fertilizers and chemical pesticides, lowering produc- tion costs and reducing environmental pollution while ensuring high yields. The poten- tial benefit of manipulating agricultural systems through modification of the rhizos- phere and phylosphere microflora is clear.

    Technical problems involved in the successful inoculation of agricultural crops in- clude the delivery of sufficient inoculum to the target, the economical production of large quantities of microorganisms, the promotion of extended shelf life, and the de- velopment of convenient formulations.

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  • 62 CRC Critical Reviews in Biotechnology

    The most important characteristics of microbial inocula are similar to those re- quested of conventional chemicals and pesticides. Alexander' has described failures to inoculate natural and already well-populated habitats with beneficial microorganisms. Goldstein et aL2 have suggested an approach that may lead to a higher rate of success in which reasons for failure are first established and then suitable organisms or prac- tices are sought. Much more research on the various microbial groups is needed to develop reliable inoculation procedures and strains and to elucidate modes of action. This review describes the accumulated knowledge, methodology, and technology for increasing crop yields by using microbial inoculants.

    11. DIRECTLY BENEFICIAL INOCULANTS

    A. Rhizobium Legume Symbioses The relationship between Rhizobium and legumes is one of the most extensively

    studied symbiotic systems and has been applied for the benefit of agriculture since the end of the last century. It is estimated that Rhizobium in symbiotic association with legumes fix about 90 x lo6 tons N per year, about twice the amount fixed annually by the chemical industry and about one half of the total amount that is fixed biologically every year. There are 16,000 to 19,000 known species of legumes in about 750 genera and hundreds of them are utilized in agriculture. Of these, most are members of the subfamily Papi l i~noideae~, and most are nodulated by rhizobia. Early studies of Rhi- zobium-legume symbiosis were extensively reviewed by Fred et al. in 1932.4 By then, most of the basic technology for inoculant production was already established. Im- provements made in the last 50 years have been mainly in the areas of selection of the more effective strains, fermentation, carrier processing, shelf life, and quality control of i n ~ c u l a n t s . ~ - ~

    In the past 20 years, there have been enormous advances in understanding the phys- iology,'O and genetics of Rhizobium, N-fixation, and n~du la t ion . '~"~ The infection p roces~ , '~ including rec~gni t ion, '~"~ has also been studied. Physiology of the nodule,20 the energy requirements of N,-fixation in the nodule," the ecology of Rhizobium in soil and the r h i ~ o s p h e r e , ~ ~ , ~ ~ and the technology for inoculant produc- tion and application in the field5.6,9 have been recently reviewed. It is expected that this accumulated knowledge will soon have an impact in legume production above that achieved so far in this century.

    1. Taxonomy The division of Rhizobium into six species according to "cross inoculation" groups,

    i.e., leguminous species mutually susceptible to nodulation by a particular kind of rhizobia, was convenient for scientists and manufacturers of inoculants for many y e a ~ s . ~ . ~ The newer classification of rhizobia, based on DNA hybridization and numer- ical taxonomy studies,'O.'' divides Rhizobium into two genera. The genus Rhizobium includes three fast-growing species, Rhizobium meliloti (Medicago), R. loti (Lupinus, Lotus), and R. leguminosarum, which is subdivided into biovar trifolli (Trifolium spp.), biovar phaseoli (Phaseolus vulgaris), and biovar viceae (Pisum, Lens). The ge- nus Bradyrhizobium contains two species, Bradyrhizobium japonicum (Glycine m a ) and Bradyrhizobium spp., which is further subdivided into Bradyrhizobium sp. (Vigna) and Bradyrhizobium sp. (Lupinus).

    2. The Infection Process and Function of the Nodule Rhizobia vary in their ability to survive in the soil, but proliferation takes place

    mainly in the rhizosphere of host and nonhost plant^.'^^^^ In some soil types, highly

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  • Volume 6, Issue 1 (1987) 63

    infective native rhizobia, which are not always effective in symbiotic N-fixation, may compete with the introduced laboratory-selected strains. This is one of the major ob- stacles to successful inoculation of legumes today. Nevertheless, it appears that only certain types of specific Rhizobium species or strains are capable of initiating an infec- tion, regardless of the composition of the rhizosphere flora. By substantially increasing the inoculum size, it is possible to cause a given rhizobial strain to be dominant and populate most of the nodules. However, it is technically difficult to reach high inocu- lum levels in the field.

    The specific ability of a given bacterial species to colonize (infect) a given plant species is still not well understood. Is Some of the compatible bacteria-plant combina- tions involving bacterial phytopathogens are highly specific and result in infection, i.e., bacterial proliferation inside the host tissue. In other cases such as tumor induction by A. tumefaciens, the relationship is nonspecific. Induction of nodule formation may be considered as a specific compatible disease (bacteria proliferate inside the plant tissue), the result of which is useful to agr icul t~re . ~ It is possible that one of the early stages of specific recognition is related to binding between plant proteins (lectins) on the surface of the root and glycoside residues on the bacterial cell envelope. The most extensively studied, documented, and reviewed system is the one involving R. trifolii and trifoliin, a lectin of white clover. Interactions between them may trigger a signal leading to the specific infection of clover root hairs by R . trif~lii.~. However, in other rhizobia, such as B. japonicum which is symbiotically associated with soybean, the connection between specificity, the early stages of binding, and mediation by the lectin- polysaccharide interaction are still poorly u n d e r s t o ~ d . ~ ~ ~ Bauer et al.I9 recently pre- sented data showing that strain 1007 of B. japonicum isolated from the field is only weakly attached to soybean roots but is as capable of nodulation, as is strain 110 which binds strongly to the roots.

    When an infective Rhizobium cell comes in contact with the root of a susceptible legume seedling, the Rhizobium proliferates and root hair colonization occurs. Sub- stances excreted by rhizobia cause curling of root hairs. The rhizobia enter the root at the base of a fold, possibly through a pore, and are then encapsulated within the infec- tion thread, embedded in a mucopolysaccharide matrix. Bacterial cells are liberated from branches of the thread into the cytoplasm of cortical cells, where they multiply, enlarge, and become pleomorphic (bacteroids). The bacteroids are located within mem- brane-bound vesicles (periplasmatic membrane) within plant cells in the nodule. Leg- hemoglobin is present within the periplasmatic membrane in contact with the bacter- oids, but it may also be encountered outside the periplasmatic membrane.*O

    Leghemoglobin facilitates the diffusion of oxygen from the nodule surface to the b a c t e r ~ i d , ~ ~ providing for efficient oxidative phosphorylation without damaging the oxygen-labile nitrogenase enzyme in the bacteroid. Current knowledge of the workings of legume nodules has been reviewed by Dilworth and Glenn.20 Carbon compounds enter the nodule cell predominantly as sugars, and C,-dicarboxylic acids are the prin- cipal sources of ATP and electrons for nitrogenase. Ammonia from N,-fixation leaves the bacteroid by diffusion along a gradient maintained by the conversion of ammonia to glutamine in the plant cytoplasm and is then transported as asparagine or allantoin and allantoic acids to the upper parts of the plant.

    For the past several years, genetic research on the development of symbiotic N- fixation has focused on the identification and mapping of genes involved in symbioses. Preliminary efforts have been made to analyze the biochemical roles of gene prod- u c t ~ . ~ ~ Rhizobium genes necessary for nodule induction, termed nod genes, are in- volved in root hair curling, infection thread growth, host range, and direct or indirect regulation of gene expression. The nod genes appear to be induced by one or more

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  • 64 CRC Critical Reviews in Biotechnology

    soluble plant factors. The nodABCD genes have been identified; they are adjacent to genes involved in N-fixation (nif A, fix A) in the nodule. There appear to be differences between the nod gene arrangement of slow- and fast-growing rhizobia.13.15

    3. Rhizobium Znoculants and Inoculation Technologies a. Rhizobium Strain Selection

    A five-stage approach (described below) for selecting highly infective and effective Rhizobium strains has been suggested by Halliday.26 Competitiveness or infectiveness implies the ability of a rhizobial strain to produce nodules in a soil containing other highly infective rhizobia. Specific recognition of host lectins possibly involved may determine infectivity. Effectiveness, or N-fixation ability, is governed by an optimal interaction between the Rhizobium and the infected legume. The nodule formed must contain many bacteroids that actively fix N. Fixation activity is governed by the amount of photosynthate reaching the bacteroids, the amount of oxygen delivered by leghemoglobin for optimal bacteroid respiration rates, and the rate of incorporation and transport by the plant of the ammonia pr~duced.~ The Rhizobium selected must be able to grow well in culture medium, in the carrier medium, and in the soil after inoculation in order to insure the formation of nodules.

    1.

    2.

    3.

    4.

    The primary characteristic used in the selection of rhizobia is the ability to no- dulate the legume crop of interest. The selection technique most commonly used involves growing the plant under sterile conditions in small containers such as glass test tubes or in growth pouches made of autoclavable plastic with an ab- sorbant paper towel insert.26 Three treatments are evaluated, one in which the plant is inoculated with the test strain and two with controls consisting of un- inoculated fertilized plants and uninoculated plants irrigated with sterile water. Plants are scored according to the presence or absence of nodules. Wacek and Brill2* have suggested measuring N-fixation by the acetylene reduction method in the test tube for early selection of effective Rhizobiumlegume combinations, especially when screening hundreds of mutants. In this stage, the objective is to evaluate the N-fixing ability of infective strains in the legume plant of interest. The most frequently utilized plant growth system for this purpose is the sterilized inverted Leonards jar in which fre- quent watering is avoided in order to reduce possible cross contamination. Plants are grown for 60 days and the following parameters are generally measured: nod- ule number, nodule fresh and dry weight, nodule color (high level of leghemoglo- bin - a red pigment correlates with effectiveness), nodule distribution, total plant fresh and dry weight, top fresh and dry weight, root fresh and dry weight, acetylene reduction rate, percentage of N in tissues, and, most significantly, the total N produced by the plant. Out of the 30 to 50 Rhizobium strains usually selected by the Leonards jar method, the 10 most effective are again tested in pot experiments for their per- formances with different soil types, pH, temperatures, fertilizer levels, water lev- els, etc. Cross contamination from adjacent pots should be avoided. Plant dry matter and total N content are the most meaningful parameters to be measured at this stage.26 Three to five strains are then evaluated under field conditions. The grain yield or the dry matter production are measured. A mid-season harvest is useful because of the many factors acting throughout the season that may affect the final yield. Precautions against cross contamination between treatments is very important.

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  • Volume 6, Issue 1 (1987) 65

    If possible, inoculation methods and types of inoculants should be evaluated at this stage. The field trial has three basic treatments, inoculation with Rhizobium, noninoculation, and noninoculation plus N fertilization.26 Before a decision can be made about widespread commercial utilization of a se- lected strain, it is necessary to test its performance in many other locations.

    5 .

    b. Types of Inoculants and Application Methods Methods for the production of Rhizobium inoculum, the various carriers for the

    cells, and methods for applying inoculants in the field have been extensively review- ed.5-9 Ideally, inoculants should contain the largest possible number of viable cells, at least 10,000 to 1 million per seed, to competitively infect the developing root following application.

    Inoculants may consist of a single strain of Rhizobium (unistrain) or contain several strains (multistrain). Legume inoculants are of two general types, those designed for application to seeds and those designed for application directly to soil. Application to soil is recommended when planting legumes in hot, dry, or highly acidic soils, when weather conditions are adverse, when seeds are coated with chemicals toxic to rhizobia, or when the natural soil Rhizobium flora competes with the introduced bacterial train.^

    Peat is the most commonly used carrier for commercial inoculants and is generally considered the most dependable.j The peat should provide a nutritive medium for the growth of rhizobia and enhance survival during distribution. In order to satisfy the requirements for a good Rhizobium carrier, peat must be highly adsorptive, be non- toxic to rhizobia, have a high organic matter content and high water-holding capacity, be easy to sterilize, and be available locally at low cost.

    Scanning electron microphotographs have shown that microcolonies of Rhizobium develop both on the surface of the particles and in crevices. The bacteria are protected from dessication within a fibrillar matrix, possibly an extracellular gum, which at- taches them to the surface of the peat particle^.'^

    In the usual method of peat-based inoculant production, rhizobia are cultured in fermenters to reach high population levels before being added to the peat. The added rhizobia (%lo9 cell/ml, 30 to 50 m l added to 70 and 50 g of dry peat, respectively) multiply further to reach maximal populations since the peat is presterilized; multipli- cation is lower in nonsterilized peat. In most successful cases, the final numbers reached are 2.5 to 3 x lo9 rhizobia cells per gram of inocu la r~ t .~~

    S o m a ~ e g a r a n ~ ~ has reported on a method of using diluted liquid cultures of Rhizo- bium and sterilized peat to increase the production capacity of inoculant manufactur- ers, particularly in locations where sophisticated high-capacity fermenters are not available. Peat is sterilized by autoclaving or gamma radiation and is aseptically mixed with a dilute Rhizobium culture. During storage for 34 weeks at 28C, the bacteria proliferate, and viable counts reach lo8 to lo9 cellslg, depending on the source of the peat. The effectiveness of the inoculants produced with diluted cultures is similar to those produced with undiluted

    c. Seed Inoculation The amount of peat supplied per kilogram of seed ranges from 4 to 6 g.9 The most

    common method of seed inoculation is the slurry method. The inoculant is first mixed with water to form a uniform pourable suspension and gums or sugars are added to improve adhesion of the inoculant to the seed. Just prior to sowing, the seeds are mixed with the slurry until thoroughly coated. Rhizobium populations rapidly decline in soil having a low pH or if the soil has been treated with fertilizers. Therefore, in

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  • 66 CRC Critical Reviews in Biotechnology

    seeds that are to be planted under these conditions, the peat layer is covered with a coat of lime to form a pellet. Preinoculated seed is seed inoculated months in ad- vance of planting before being offered for sale by seed processing companies. Results achieved with preinoculated seed have not been reliable.

    Inoculants can also be applied directly to the soil. Soil inoculants may be in the form of liquid or frozen concentrates distributed by drip or spray systems, or inoculated granules in which small marble, calcite, or silica grains are wetted with an adhesive and mixed with a peat powder-type inoculum. After drying, the granules are broadcast over the field. In the natural peat granule method, Rhizobium is added to natural peat granules ranging from 300 to 800 pm in diameter. Moisture is added to permit growth and multiplication of the rhizobia in the granules. The inoculant is distributed uni- formly using a granular applicator drill atta~hment.~.

    Other inoculant preparations in use include oil-dried preparations of talc or vermi- culite and rhizobia entrapped in polyacrylamides, alginates, or x a n t h a n e ~ . ~ ~ These biopolymers are remarkable for their rheological properties (viscosity) which limit heat transfer when inocula are spray dried. They are very stable when stored dry and recover their viscosity immediately after being applied to soil. In the field, the microorganisms are protected until the polymer structure has been totally degraded. The polymers are nontoxic to R h i z o b i ~ m . ~ ~ In France, where polysaccharide granules have been applied to soil, only a high rate of 26 to 54 kg/ha produced a significant increase in yield over seed-applied inoculants.

    B. Frankia: Inoculants for Forestry 1 . Economical Importance, Organisms Involved, and Symbiosis

    In order to meet future wood demands, an intensive program of reforestation is needed.33 Reduction of forest areas at annual rates varying from 7.5 to 20 million ha is the result of logging, mining, and shifting c~ l t iva t ion .~~ Since high-quality soils tend to be utilized for agriculture, areas of low fertility remain for reforestation.

    Plants capable of forming root nodules for N-fixation in association with the fila- mentous bacterium Frankia (actinomycetes) have been found worldwide in 23 genera belonging to 8 families. Among these are more than 200 species of dicotyledonous woody specie^.^^-"^ The root nodules fix N and cause it to accumulate in the soil at rates equivalent to those achieved by herbaceous legumes. Estimates of the N fixed annually range between 50 to 250 kg N per hectare, depending on the plant species and the region.34

    The potential role of Alder (Alnus species) in intensive temperate-region forestry has been recognized. Alders are large trees and can be used for timber production. N-fixing trees are used as fuel, forage, and green manure in the tropics. Casuarina is the most important actinorhizal plant of the tropics; the genus contains well over 60 species.37 Myrica gale, an actinorhizal shrub capable of fixing substantial amounts of N, is widely distributed in wet lands and along shores in the northern U.S., Canada, and E u r ~ p e . ~

    Ultrastructural studies of actinorhizal Casuarina, Alnus, Myrica, and Comptonia have shown that infection takes place via root hairs and is followed by invasion of the root cortex and the proliferation of multilobed, modified, lateral root branches that form typical nodules with nodule rook3 The endophyte within the root hair forms filaments surrounded or encapsulated by a polysaccharide capsule produced by the host cytoplasm. The filaments invade the cortical cells, dissolving middle lamellae and cell walls, and often forming strands of invading filaments. Filaments are 1 pm in diameter, separated, and branched. They ramify throughout the cells of the root cortex and cause cell proliferation and cell hypertrophy. This results in the formation of a

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  • Volume 6, Issue 1 (1987) 67

    cluster of swollen nodule lobes at each infection site with nodule roots. The nodule roots remain uninfe~ted.~ ' There are two general types of nodules known, the Alnus type and the Myrica type.35 Comparative studies of root nodules of different actinor- hizal species show that the vegetative filaments, sporangia, and vesicles of Frankia within the nodule assume various arrangements, forms, and structures depending on the host.39

    Isolated Frankia are able to infect a wide range of plant species. An isolate from Cornptonia, for example, was capable of nodulating plants of both the A h u s and the Myrica groups, but not Casuarina. The cross-inoculation groups are therefore much broader than with R h i z o b i ~ r n . ~ ~

    Frankia strains have been isolated in pure culture. In vitro, they produce vegetative hyphae, sporangiospores within sporangia, and vesicles in which N2-fixation takes place. In Frankia from Casuarina, a fourth type of structure, i.e., reproductive toru- lose hyphae which may play an important role in survival and reproduction of Frankia, is found."O

    2. Preparation of Frankia Znoculants Before in vitro cultures of Frankia spp. were accomplished, isolates were obtained

    from nodule homogenates and were used to nodulate plants produced for studies or for use in the field.

    Fresh nodules taken from trees in the field were homogenized with a blender and washed several times by repeated centrifugations with phosphate buffer saline contain- ing 2% of polyvinylpyrrolidone (PVP) for elimination of phen01.~' The crushed nodule suspension was diluted in tap water and sprayed directly on plants; 1 to 2 g of nodule fresh weight was sufficient for 1000 plants.

    The seedlings were then watered to cause percolation of the crushed nodule con- t e n t ~ . ~ ' Since the isolation of numerous Frankia strains, in particular from Afnus and related species and more recently from Casuarina species, there have been advances in the production of inoculants on an industrial scale.41 The use of pure cultures of Fran- kia permits strict control of the microbiological components of the inoculum. Pure culture inoculant was found to be superior to crushed nodule homogenates in yielding reproducible nodulation of seedlings.

    The recent availability of in vitro propagated Afnus clones will provide opportunities for selecting the plants and for control of the genotype of the selected symbiont (Fran- kia) being tested.42

    Pure cultures of Frankia are obtained by surface sterilization of the root nodule for removal of contaminating organisms and by the use of microdissection techniques to remove the internal tissues which are then incubated in a suitable culture medium to reach high cell density. Highly concentrated inoculum is necessary for successful inoc- lat ti on.^^ Lalonde et al.44 were successful by using a 3% aqueous solution of osmium tetroxide for surface sterilizing the nodule for 30 sec to 6 min and then washing several times with sterile water to remove the osmium tetroxide fixative. The treated nodules were sliced under sterile conditions in the presence of a P V P buffer solution. The pieces of sliced nodules with exposed endophyte protected from phenols by the PVP were aseptically transferred to glass tubes containing growth medium with glucose and leci- thin.44 Propionate is also used as a sole organic carbon source with casamino acids serving as a source of N.45 Typical Frankia colonies develop at the periphery of the blackened piece of nodule. For N2-fixing vesicle formation, the same medium is used without combined N.45 The actinorhizal trees Alnus and Casuarina are currently pro- duced in commercial nurseries for outplanting for forest regeneration, mine spoil re- clamation, land stabilization, and biomass production. There is a need for well-nodu-

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  • 68 CR C Critical Reviews in Biotechnology

    lated healthy seedlings for transplanting to the often poor soil of these areas. Plants grown in containers are inoculated by selected effective Frankia strains.

    Suspensions of pure cultures of Frankia are applied directly in the containers as a soil drench at the time of seeding or as a root dip for seedlings transplanted into con- tainers. In Canada, successful inoculation with pure cultures of Frankia was achieved either by soil injection or by spraying with greenhouse watering device^.^' Factors that may contribute to the successful establishment of N-fixing nodules are the quantity and quality of the Frankia inoculant, time and method of inoculation, and nutritional status of the host plant.46 Production in containers is particularly advantageous, as soil mixes are fumigated and sterilized allowing for nodulation of the seedling with the selected Frankia inoculum. Optimal plant growth conditions such as peat-vermiculite at soil pH between 5.5 to 8.8 can be maintained.46 The best results with red alder were obtained in plants inoculated at planting and not after 4 weeks of growth. Plants fer- tilized with dilute mineral solutions without N showed the best nodulation and growth. Addition of N to the mineral solution promoted plant growth but inhibited nodula- t i ~ n . ~ ~ For preparation of inoculum, pure cultures of Frankia are first washed with an N-free mineral solution and then homogenized by sonication. In this process, 1 I of concentrated inoculant in N-free mineral solution was enough for the successful no- dulation of 100,OOO seedlings. The Frankia stock inoculant can be stored at 4C until ~tilization.~

    Actinorhizal plants have already been inoculated with Frankia on a large scale. From 1979 to 1984, more than seven million inoculated seedlings, mainly of Alnusand Elaen- gus, were produced in Canada and used for land reclamation in northern Quebec and by the City of M ~ n t r e a l . ~ ~

    C. Plant-Growth-Promoting N-Fixing Bacteria Since the first part of the 20th century, there have been attempts made to improve

    yields of plants by inoculation with bacteria capable of fixing atmospheric N after colonizing the rhizosphere of nonleguminous plants, mainly forage and grain grasses. An extensive program of bacterization with N2-fixing bacteria of the genus Azotobac- ter (A. chroococcum) was carried out, mainly in the U.S.S.R., in the 1 9 5 0 ~ ~ About one third of the field inoculation experiments showed improvements of 8 to 12% in yields. The work on Azotobacter as an inoculant was reviewed by B r ~ w n , ~ ~ . ~ ~ who concluded that the slight improvement in plant growth obtained in soils with added mineral fertilizers was derived from processes other than biological N-fixation. Plant- growth-regulating substances may be produced by the bacterium in the root zone, and in some cases there were indications of biological control of plant pathogen^.^' Dob- ereiner et al. described a specific N-fixing association between Azotobacter paspali and the rhizosphere of Paspalum notatum cv. Batatais, a tetraploid, but not in cv. Pensacola, a diploid plant. It was further demonstrated by the N-15 dilution method that about 10% of the accumulated N in cv. Batatais was derived from biological N- fixation. However, Barea and BrownSZ suggested that plant-growth-promoting sub- stances produced by A. paspali caused the growth-promoting effects of Paspalum. Dobereiner and Days3 isolated and described N-fixing bacteria of the genus Azospiril- lum living in close association with the rhizosphere of grain grasses such as corn, sorghum, wheat Setaria, Panicum, Digitaria, and Pennisetum. The genus Azospirillum has been the subject of extensive research and re vie^.^^-^^ Azospirilla are highly motile organisms with a polar flagellum utilized for swimming and peritrichous flagella with a shorter wavelength utilized for swarming on semisolid The cell accumu- lates poly-/3-hydroxybutyrate granules which are utilized as a source of carbon and energy during starvatiod4 and produce cysts for survival under unfavorable condi- t i on~ .~ In suspension, clumps are formed. Azospirilla are capable of utilizing a wide

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    variety of carbon and N sources for g r ~ w t h . ' ~ They adapt to aerobic, microaerobic, and anaerobic conditions and move aerotactically along self-created gradients of low dissolved oxygen tension.66 They chemotactically follow gradients of chemoattractants excreted by root^.^^.^^ All of these physiological properties provide the cells with mech- anisms for colonization, proliferation, and survival in the highly competitive rhizos- phere environment.60

    Extensive experimentation has demonstrated that N-fixation activities take place in plants (Setaria, sorghum, wheat) inoculated with A z o ~ p i r i l l u m , ~ ~ ~ ~ ~ 6o but the rates ob- served were very low when compared to those of the legume-Rhizobium symbioses and could not account for the observed N-yield increases in Azospirilluminoculated plants. 58 .60 .62 It has therefore been concluded that the beneficial effects of Azospirillum on yields are caused, as in A z o t o b a ~ t e r , ~ ~ mainly by mechanisms other than biological N-fixation. In the case of Azospirillum, the plant-growth-promoting effects in both the greenhouse and the field have been much more consistent than those reported for Azo- tobacter. 6o

    Recent work has shown that the inoculation of wheat, sorghum, and corn plants with an inoculum size of lo6 to lo7 Azospirillum cells per plant has a marked effect on root tip morphology, proliferation of root hairs, root surface area, root branching, and the general development of the root ~ y s t e m . ~ ~ . ~ ' Interestingly, increased root growth promotes rather than inhibits shoot growth in Azospirillum-inoculated plants. Inoculum concentrations of less than lo5 Azospirillum cells cause only slight effects on root growth, where with a very high number of cells lo8 to 1O1O, an inhibition of plant growth is observed in laboratory-grown seedlings.61 The positive effects on root devel- opment cause an increase in mineral and water uptake from the soil. Minerals and water accumulate at higher rates in plant parts, leading to increased yield.62 The mech- anisms by which Azospirillum affect roots are not known. In culture, the organism produces plant-growth-promoting substances such as indole acetic acid and cyto- kin in^.^^ Although some reports imply that there may be specific Azospirillum species or strains with affinities for a specific type of plant,70 most of the evidence shows a more generalized effect of a given Azospirillum species on several species and cultivars of plants, provided the plants are inoculated with an optimal concentration of bacterial cells.62

    1. Inoculation in the Field with Azospirillurn So far, inoculation methods utilized for Azospirillum have been based on technology

    developed for Rhizobium inoculant preparation and application. The best results have been obtained by dripping peat suspensions containing lo9 Azospirillum cells per gram of peat into the furrow at a rate of 1 to 2 kg/ha or by spreading granular-peat inoculant at the time of sowing at a rate of 4 to 6 kg/ha in soils fertilized with optimal levels of N, P, and K.62

    In field experiments, inoculation with Azospirillum led to 10 to 30% increases in grain and forage yields of wheat, sorghum, and corn over noninoculated controls. A rough estimate of efforts throughout the world shows a 65% success rate, assuming that both positive and negative results have been r e p ~ r t e d . ~ ~ . ~ * It must be stressed that the concentration of Azospirillum cells needed in the inoculum for colonizing roots and affecting root development may be dependent on soil microbial composition, or- ganic matter content, and the total number of microorganisms competing for coloni- zation sites on the roots. This makes the development of inoculants suitable for all soil types more difficult and may explain past failures in work with Azospirillum.

    Based on our current knowledge, it seems that the use of Azospirillum inoculant on a commercial scale for improving yields shows potential for fields moderately fertilized with N, P, and K. In developed countries, the farmer may obtain yields even higher

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    than those achieved in fully fertilized fields. In less developed countries where less fertilizer is commonly used and, in many cases, less water is available, Azospirillum will insure higher yields, but the fields will need to be fertilized for the following sea- son. In the near future, widespread utilization of Azospirillum inocula will be possible mainly in soils comparable to those found in Israel, in which low organic matter con- tent allows the introduced bacterium to compete for colonization sites on the roots.

    D . Vesicular-Arbuscular Mycorrhizae The vesicular-arbuscular mycorrhizal (VAM) fungi are beneficial symbiotic micro-

    organisms that increase the growth and yield of most plants by improving P uptake, resistance to drought and salinity, and tolerance to pathogen^.^'-^^

    The anatomical features of VAM infections have been described by Brown and King.75 The presence of vesicles and arbuscules is, by definition, the diagnostic crite- rion for VAM-infected roots. Vesicles, which probably function as storage organs, are usually oval and are located between and sometimes within the cortical cells. Arbus- cules are analogous to the haustoria of obligate parasitic fungi and function in the bidirectional transfer of carbohydrates from the plant to the fungus and minerals, such as phosphate from the fungus to the plant. Some common genera of VAM fungi are Acaulospora, Entrophospora, Gigaspora, Glomus, and Sclerocystis.76 (They differ from other fungi in the morphology of structures produced exterior to the root such as chlamydospores, sporocarps, and azygospores.)

    VAM are found in most agronomic plant species, including pasture and forage leg- umes, fruit trees, corn, wheat, barley, potatoes, and many other vegetable^.^^ The potential benefits of VAM inoculation for field crops have been reviewed exten- ~ i v e l y . ~ ~ , ~ ~ In order to fulfill this potential, large-scale production of VAM inoculum is essential. Currently, since the fungi cannot be cultured in the absence of a living root, the only way to produce suitable quantities of VAM inoculum is on roots of susceptible host plant^.'^.^?.^^ This limits the commercial application and distribution of large quantities of specific, highly infective, uncontaminated inoculum.

    The current procedure for inoculum production was described by Menge and Tim- mer in 1982.78 The inoculum consists of a host-plant growth medium such as soil, vermiculite or peat, and host roots associated with VAM hyphae and spores which have been ground and dried. These media must be sterilized in order to eliminate path- ogens and then inoculated with spores or infested roots. Sudan grass (Sorghum vul- garae) is frequently used for soil inoculum production. After the VAM is established on the host roots, it is applied to the seedlings that will be transplanted in the field. This inoculum is very efficient and its use is practical for nursery inoculation, but it has several severe limitations as far as commercial application is concerned. Among these limitations are its bulk and the effort involved in processing, sterilization, pack- aging, and distrib~tion.~

    A possible breakthrough in VAM inoculum production is the use of the nutrient film technique (NFT). In this method, plants are grown in a shallow layer of rapidly flowing nutrient solution. The root mats produced by NFT-grown plants seem ideal for the production of an inoculum which is more easily harvested, more concentrated, and less bulky than that produced in soil described an NFT apparatus adapted for VAM production. VAM infections were produced in bean plants (Phaseolus vulgaris), and the infected roots were used as an efficient inoculum for corn and bean seedlings grown in sterilized soil. In a similar study, Elmes and Mosse80 demonstrated the possibility of producing Glomus sp. VAM on corn roots (Zea mays) using NFT. Elmes et aL81 extended these studies by demonstrating the effectiveness of NFT-grown mycorrhizal bean roots as inoculum under field condi- tions. They suggest that 60 kg of NFT-produced inoculum is equivalent to 2500 kg of

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  • Volume 6, Issue 1 (1987) 71

    soil-produced inoculum, the amount required to inoculate 1 ha. This quantity can be produced in a 200-m NFT The NFT system therefore has practical implica- tions for mycorrhizal inoculum production.

    Once produced, VAM inoculum must be processed and applied. In order to over- come the problems involved in application of large amounts of inoculum, methods have been developed to separate and concentrate the VAM spores from soil by wet- sieving elution or centrifugation. 74.77.82.83

    Both spores and root inocula can be applied by spore drill,84 seed c ~ a t i n g , ~ . ~ ~ or seed pelleting. Seeds were pelleted with oil infected with Glornus sp.85.86 or methyl cellulose and ground prelite mixed with Glomus rnicrocarpus.82 Sowing these pelleted seeds resulted in an increased growth of onions, tomatoes,82 rye grass, and c10ver.~ This technique could thus be a suitable method for introducing VAM to soils.

    Methods in which the VAM inoculum is localized are superior to traditional broad- cast applications in that they require significantly less inoculum.

    The importance of VAM is beyond doubt and its application to soil is beneficial to many crops. In particular, VAM are beneficial in soils where insoluble (immobile) essential elements (like P) are in short supply. Since it must be grown on host plant roots, methods for achieving high infection rates are of great importance. Of the dif- ferent systems described, the NFT system looks the most promising.

    E. Ectomycorrhiza The Ectomycorrhizae include fungi belonging to many genera and are most common

    among forest and ornamental tree^.^^.^' They are of major importance in forest regen- eration. The growth of these fungal symbionts is stimulated by root exudates. Hyphae grow over the surface of host feeder roots, forming a fungal mantle, which replaces the root hairs with fungal strands, greatly increasing root surface absorbtive area. Hy- phae then develop around root cortical cells and form an interconnecting network known as the Hartig net. Most ectomycorrhizal fungi produce sporophores that can be used for inoculation as well as soil inoculum. However, the use of pure mycelial cultures of selected fungi for inoculation is the most biologically sound method since pathogens and other contaminants are e x ~ l u d e d . ~ , ~ ~

    Large-scale nursery application of vegetative inoculum has been severely hampered by a shortage of inoculum. MoserE9 was the first to develop a method of growing vegetative inoculum of ectomycorrhizal fungi using Suillus plorans. He grew the fun- gus in an aerated liquid medium for 3 to 4 months and then transferred it to sterilized peat moss for another 2 to 4 months. He tried to apply agar mycelial inoculum or mycelial suspensions, but found them ineffective in the nursery. Various groups have successfully followed Mosers example, with some modifications, in Argentina, Can- ada, Australia, and the U.S. The development of vegetative inoculum of Pisolithus tinctorius by Marx and co-workers is an excellent example. P. tinctorius was chosen by this group because of its broad host range, rapid growth in pure culture, and its value in the reclamation of marsh areas by the planting of pine^.'^.^^

    A medium consisting of vermiculite and 5 to 10% peat by volume moistened with salts and glucose nutrient medium proved excellent for growing mycelial inoculum, which is superior to basidiospore inoculum in infecting pine roots. Other organic sub- strates such as peanut hulls, corncobs, or pine bark are not suitable because they re- lease fungal-growth inhibitors during a u t ~ c l a v i n g . ~ ~ A commercial formulation of my- celial inoculum of P. tinctorius, which is grown on vermiculite peat medium, has been developed by Abbott L a b o r a t o r i e ~ ~ ~ ~ ~ under the trademark MycoRize B . The use of peat in this formation is important since it has a strong buffering capacity, keeping the pH below 6; this was not possible using chemical buffers.90 The use of peat may have additional positive effects. P. tinctoriusis able to produce fulvic and humic acids

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    in pure culture, and vegetative growth of the fungus is stimulated by fulvic acid.9' In the culture medium, peat may furnish essential humic acids or their precursors to P. tinctorius, much as organic matter does in forest soil. Thus, peat may play a vital role not only in fungal growth but also in the production of effective i n o ~ u l u m . ~ ~

    111. INDIRECTLY BENEFICIAL INOCULANTS

    A. Systems for Biological Control of Plant Pathogens 1. Fungal Antagonists

    Biological control of soil-borne plant pathogens by the application of microor- ganisms to soil is a potential nonchemical method of plant disease involving mechanisms such as direct antagonism, competition, hypovirulence, and cross protec- tion. Several fungi, such as Gliocladium, Penicillium, and Caetomium, and members of the genus Trichoderma are antagonistic to plant pathogenic f ~ n g i . ~ ~ , ~ ~ A mycopar- asitic mechanism is involved in the antagonistic relationship between Trichoderrna and fungi such as Rhizoctonia solani and Sclerotium rolfsii. This is a complex process involving several steps. First, the mycoparasite grows directly towards its host in a chemotropic manner.95 When the antagonist reaches the host, it coils around its hy-

    A recent study indicates that specific recognition between Trichoderma sp. and S. rolfsii is mediated by le~tins.~' The antagonist is able to produce cell wall- degrading enzymes such as chitinase and glucanase, thus penetrating the cells of its host.98 Degradation and penetration of the host cells were observed by electron mi- c r o ~ c o p y . ~ ~ . ~ ~ Trichoderma spp. have been very widely tested for control of many path- ogenic fungi. 93.94.100- 106 Direct inoculation of antagonists to soil is not, however, eco- nomically feasible.93 Wells et al.lo7 were the first to report field control of S. rolfsii by infestation of the soil with Trichoderma harzianurn grown on an autoclaved mixture of ryegrass and soil. Rather than trying to alter the soil microflora, they overwhelmed the infection court with T. harzianum and its food base, thus reducing disease inci- dence.

    Since then, many workers have followed this method of growing an antagonist on an organic substrate which serves as both a delivery system and a food base. Sand-

    and lignite-stillage carrier"' have all been employed as carriers. Wheat bran has been suc- cessfully used as a food base for T. harzianum.101-103 The wheat bran preparation proved superior to a directly applied T. harzianum conidial suspension in its activity against S. rolfsiiand in its ability to survive in soil.'O' The release of cell wall-degrading enzymes is apparently the mechanism by which T. harzianurn acts as an antagonist. High enzymatic activity was detected in the wheat bran culture filtrate.98 During fun- gal growth, however,'pH levels increased to pH 8, probably due to the release of am- monia from proteins. The addition of peat (50% by volume) effectively stabilized the pH level at 5.5 without decreasing the Trichoderma population density and was bene- ficial in preventing bacterial contaminations. '05 Moreover, the survival of Trichoderrna was better when it was grown on this mixture than on wheat bran alone. It seems that the use of a proper food base is a critical factor in determining the success or failure of directly applied soil biocontrol agents in controlling soil-borne plant pathogens. Application of a food base with the biocontrol agent might overcome fungistasis and enable the introduced organism to grow, colonize the soil, and degrade propagules of plant pathogenic fungi. Baker et a1."* also discussed the beneficial effects of peat-bran culture for the formulation of Trichoderma.

    Although promising, introduction of Trichoderma preparations to soil requires large amounts of inoculum. Application of the antagonist to the seed has been suggested as an alternative approach for controlling damping-off diseases caused by Rhizoctonia

    barley grain,Io9 wheat straw,"O diatomaceous earth and

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    and Pythium . I I 3 . l l4 T . harzianum was also applied to the rooting mixture of Chrysan- themum, resulting in a significant increase in growth response and beneficial effects even after transplanting, thus making application of Trichoderma more cost effec- tive. 115

    In all of the studies described above, the antagonist was produced by fermentation on solid substrates. Papavizas et a1.Il6 have recently shown that it is possible to use liquid media to produce viable inocula of Trichoderma and Gliocladium in a deep tank fermentation system. The fungi developed large amounts of biomass containing my- celia, chlamydospores, and some conidia. It was demonstrated that fermentor biomass preparation formulated as a powder slurry or alginate pellets and added to soil prolif- erated and suppressed diseases. *I6 Papavizas9' suggested that the development of a wettable powder formulation composed of dry fine fermentation biomass and proper wetting and suspending agents should not be difficult.

    Trichoderma mutants resistant to pesticides have been produced by exposure to fun- gicides and mutagen^.^^,^^.^^^ A few isolates tolerant to benomyl showed an enhanced ability to suppress plant pathogen^.'^^ However, other techniques for genetic manipu- lation, such as protoplast fusion and transformation, have not been applied yet to fungal biocontrol agents. More understanding of the mechanisms involved in biocon- trol at the molecular and biochemical level will lead to improvement and the ability to predict antagonistic capabilities.

    2. Biological Control of Crown Gall Agrobacterium tumefaciens, the causative agent of crown gall, is a major pathogen

    causing damage to many crops throughout the world. L 1 8 - 1 2 0 Crown gall diseases have often been controlled by a technique developed by KerrZ2l

    which involves preinoculating plants with A . radiobacter strain 84.118,122 Strain 84 pro- duces a bacteriocin - Agrocin 84 - with a very specific host range.

    There is no generally accepted definition or classification of bac te r ioc in~. '~~ They are compounds of low or high molecular weight with antibiotic-like effects on bacterial strains belonging to the same or related species as the producer. Thus, bacteriocins possess a high level of biological specificity, separating them from the broad-spectrum antibiotics .Iz3

    Agrocin 84, a low molecular weight adenine nucleotide-like compound, inhibits the growth of A. tumefaciens carrying only a nopaline-type Ti-plasmid. Moreover, it is not effective against biotype-3 strains that cause crown gall in grapevines.12o

    Another possibility for control of crown gall by A. radiobacter resides in the com- petition for sites for A. tumefaciens transfer of the T-DNA fraction of the Ti-plasmid to the plant cell. Thus, for nopaline-type crown gall control, there may be a combina- tion of agrocin production and competition for binding sites occurring in A. radiob- acter strain 84.I2O

    The standard method for treatment of plants with A. radiobacter is to dip cuttings of rooted plants in a suspension of lo7 to lo9 cells/ml immediately before planting.122

    Efficient biological control of crown gall (nopaline type only) by strain 84 has been reported from a number of countries.122 In most cases, control has been 80 to 100% successful.

    It is possible that biological control could break down in the field by the transfer of a Ti-plasmid from pathogenic A. tumefaciens to A. radiobacter strain 84. It is there- fore necessary to develop strains for biological control that are deficient in plasmid transfer and mobilization functions. It is also necessary to develop strains capable of controlling any of the nopaline octopine and agropine Ti-plasmid-encoded A. tume- faciens strains. lZo

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    3. Biological Control of Frost Injury Frost injury to plants is a severe problem in agriculture, causing heavy yield losses.

    Ice nucleation by active strains of Pseudomonas syringaeand Erwinia herbicola, which reside as ephiphytes on the leaf surface, initiate ice formation and thus cause frost injury to plants. In the field, frost injury has been reduced following reductions in the ice nucleation activity of these bacteria.lz4 Lindow et a1.124,126 have shown that the frost sensitivity of corn plants is reduced when plants are treated with antagonistic non-ice- nucleating bacteria. An isolate of E. herbicola was used for growth chamber and field experiments. The bacteria were grown on nutrient agar containing 2.5% glycerol, har- vested, suspended in phosphate buffer (pH 7) or nutrient broth, and sprayed at rates of 0.5 m l per plant. The effective bacterial concentration was 3 x 10' colony-forming units per milliliter.1z5~126

    More studies have revealed that the mechanism of antagonism in this system is the direct competition for growth sites and/or nutrients at these sites.lz7 It was thus con- cluded that a mutant of the pathogenic bacteria not active in ice nucleation would be the best competitor. Orser et al. 128,129 used recombinant plasmids with well-character- ized deletions which removed a portion of the ice gene sequence to incorporate specific deletion in the genome of P. syringae. These genetically engineered deletion strains no longer incited frost injury to plants in growth chamber experiment^.'^^ This could be a pioneering system for the use of genetic engineering for the improvement of biocontrol agents. However, field trials for establishing the efficacy and biological safety of these strains as control agents of frost injury have not been performed because of a prohi- bition by the US. The tests were disallowed because of concern about poten- tial ecological hazards involved in releasing genetically engineered organisms into the environment.

    4. Plan t-Gro wth -Promoting Fluorescent Pseudom onads Introduction of selected antagonistic fluorescent pseudomonads to the rhizosphere

    has been shown to suppress harmful microbial a~ t iv i ty . ' ~ ' . ' ~~ Substantial yield increases may possibly be obtained by using these bacteria, either directly by plant-growth-pro- moting e f fec t~"~ or indirectly by protecting plant roots from the many potentially del- eterious microorganisms present in agricultural soil^.'^^.'^^ In one example, a history of monoculture of a wheat crop susceptible to the specific soil-borne fungal pathogen Gaumannomyces graminis var. tritici causing take-all disease in Washington State is usually a prerequisite for the induction of suppressiveness towards the pathogen. 135 It is suspected that suppressiveness (soil not conducive to a particular disease) is due mainly to the development of antagonistic pseudomonads. On the other hand, the activity of deleterious rhizosphere microorganisms (generally unidentified) other than specific soil-borne pathogens is apparently increased as a consequence of certain cul- tural practices, such as high-cropping frequencies of the potato in Holland, and, in some cases, may cause severe yield r edu~ t ion . '~~

    Another example is the successful control of Fusarium wilt by introduction of Pseu- domonas from suppressive to infested

    Fluorescent pseudomonads possess a versatile metabolism for utilization of sub- strates released by roots, short generation times, mobility, and capability to colonize roots. They produce a wide variety of secondary metabolites antagonistic to other mi- croorganisms.'31-133.13s Rhizobacteria must effectively colonize the root surface in or- der to have a physiological effect on plant

    Plant-growth-promoting rhizobacteria (PGPR)13' have been obtained mainly by se- lecting Pseudomonas colonies that strongly inhibited the growth of a variety of path- ogenic and saprophytic rhizosphere fungi and bacteria in culture.

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  • Volume 6 , Issue 1 (1987) 75

    One hypothesis on how rhizobacteria promote plant growth states that the aggressive colonization of the root system results in displacement or exclusion of deleterious com- ponents of the microflora; this is generally correlated with an overall decrease in the number of rhizosphere microorganisms (fungi and bacteria) present. 131-134,136 An im- portant mechanism seems to be competition for iron (Fe) by the release of siderop- hores, i.e., secondary metabolites with a strong affinity for Fe3+.133.136 Selected mu- tantsI3' and mutants obtained by transposon mutagenesis which have lost the ability to produce siderophores in vitro also lose their plant-growth-stimulating properties, al- though they colonize roots as well as normal However, it has not been dem- onstrated that production of siderophores takes place in the rhizosphere. 138

    Another hypothesis suggests that the production of antibiotics by PGPR inhibits growth of deleterious microorganisms. Mutants that do not produce antibiotics have been seen to colonize roots at densities similar to those typical of wild strains, but the latter failed to increase yields of potatoes in the field.'"O Another possibility is that PGPR produces plant-growth-promoting substances. However, in the case of PGPR, effects on plant growth have been generally obtained only in the presence of microor- ganisms (minor pathogens, pathogens) where promotion by Azospirillum is also ob- served under gnotobiotic conditions in the absence of harmful rhizosphere micro- flora.60

    Direct plant-growth-promoting effects have been observed recently in soybeans and canola (rape seed) under gnotobiotic conditions in plants inoculated with some isolates of Arthrobacter, Bacillus, Enterobacter, Serratia, and Pseudomonas. 173

    Significant increases in yield by seed and tuber bacterization have been demonstrated in field experiments with sugar beets, potato radish, and other crops136,137.141 and with canola and soybeans.173

    The most significant data on growth stimulation have been obtained in pot experi- ments where the development of roots or tubers (potatoes, sugar beets) was measured long before Impressive increases in field yields were obtained in crops hav- ing a relatively short growing season and a relatively restricted root volume, such as the radish. According to Schippers et al.,134 significant growth promotion by PGPR treatment takes place only in soil with a history of high-cropping frequency of a par- ticular crop, such as potatoes in Holland.

    In many cases, however, no significant increases were observed. These results have been attributed to variability in the field and to inadequate colonization of roots by the introduced PGPR.I3"

    Inoculation of seeds with aqueous bacterial suspensions was not a satisfactory method because of the low survival of PGPR after drying. 136.137 Successful inoculation has been obtained by pelleting seeds with bacteria mixed with preservatives and adhe- sive substances. Cellulose methyl ether has proven the best preservative for a wet pel- leting formulation, and xanthan gum is best for a dry powder f o r m u l a t i ~ n . ' ~ ~ . ~ ~ ~ Coat- ing sugar beet seeds with screened peat, talc, or diatomaceous earth provided good adhesive ability without affecting seed germination. Dosage-response studies have in- dicated that the minimum number of usable cells needed for uniform colonization and growth promotion are lo5 PGPR cells per seed or lo7 cells per gram dry i n ~ c u l u m . ' ~ ~

    B. Fungi as Biocontrol Agents of Arthropods Insects and mites are major pests in agriculture, causing great yield losses in all

    crops. Interest in the utilization of microorganisms pathogenic to insects as bioinsecti- cides is largely a result of the problems associated with the use of chemical pesti- cides. 142,143 Chemical insecticides may cause environmental damage and kill beneficial insects. Moreover, insects may acquire resistance to the chemicals, leading to the de- velopment of new pest problems.

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    More than 400 species of fungi can attack insects and mites.144 Their taxonomy, modes of infection, and host ranges have been investigated and reviewed in de-

    Examples of fungi frequently used for biocontrol are Verticillium lecanii for the control of aphids, Beauveria bassiana for the control of the Colorado potato beetle, Hirsutella thompsonii for controlling mites, and members of the entomophtorales which cause disease in a wide variety of insects.

    The first step in the development of a microbial insecticide is mass production of its infectious propagules. The two methods used are culture on solid substrate and liquid fermentation. Cereal grains are the most attractive solid substrates as they are uniform, readily available, inexpensive, nutritious, and easy to sterilize and process. Solid fer- mentation takes place in trays, bags, or rotating drums. In all these methods, a long fermentation period (10 to 14 days) is required, and the maintenance of sterile condi- tions is problematic.45.148

    Despite limitations, solid-state fermentation is the only way to propagate some spe- cies, such as most strains of H. thompsonii, which do not sporulate in liquid culture.149

    Liquid fermentation in submerged culture offers better control of the fungal growth than solid fermentation. Temperature, aeration, agitation, pH , and nutrient concen- tration can be altered during growth, allowing for harvest of cultures precisely at the morphological or physiological stage optimal for processing and ~ t 0 r a g e . l ~ ~ V. lecanii is produced by liquid fermentation, yielding 1 O O spores/ml. 148

    Other important steps in the production of biological insecticides are processing, formulation, and development of delivery systems. 149.150

    Several commercial preparations of fungal insecticides have been produced. V. le- canii was the first fungus to be registered. Verta1ecQ is used in commercial glass houses for control of aphids. V. lecanii blastospores are less stable than conidia and must be distributed for application immediately after culturing. 145~148 Abbott Labora- tories has developed a bioacaricide called Mycon 8, a wettable powder formulation of spores and hyphae of H. thompsonii.

    BoverinB is produced in the U.S.S.R. by culturing B. bassiana in a two-stage process. First, the biomass is produced as mycelium in a fermentor. This mycelium is used to inoculate surface cultures in trays for sporulation. 148,151

    While the use of fungal bioinsecticides seems promising, a major drawback is their poor storage c a p a c i t ~ . ~ ~ . ~ ~ More basic research needs to be done on storage problems; a better understanding of the molecular basis of survival may produce genetic solutions to this problem. 146.148 Genetic manipulation may also be useful for combining favora- ble characteristics of different strains, thus providing a high-yielding, easily stored, potent strain effective under a wide range of temperatures and humidity. L48 Genetic improvement could be achieved by traditional mutation or protoplast fusion. Proto- plasts have already been produced from B. ba~s iana . ~~

    tail. 142. 145 - 147

    C. Biological Control of Weeds with Plant Pathogens Conventional chemical weed control technology has proven inefficient in many

    cases. An alternative to the herbicides may be found in fungal plant pathogens. This approach has been adopted only recently; significant progress in the use of pathogens for weed control and for supplementing existing weed control technologies has been made only during the last decade.Is3 Research concerning the use of fungal plant path- ogens for weed control has recently been r e ~ i e w e d . ~ ~ * - ~ ~ There are two common con- trol approaches: (1) the classical tactic which implies importation of a foreign path- ogen from the area of coevolution with its host and its release into a new geographic area where the host has become a problem and (2) the bioherbicide tactic which

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    involves mass production of a potential pathogen and its application as an herbicide. The second approach includes the preparation of microbial inoculants.

    Quimby and Walker,lS7 describing the characteristics required of a bioherbicide, state that it should be genetically stable, have a restricted host range, produce suitable inoculum in artificial culture, and cause disease under a wide range of environmental conditions. Native pathogens are favored over introduced exotic ones.

    A number of successful attempts at microbial weed control have been reported. Templeton et al. 160 used Colletotrichum gloeosporioides to control weeds in rice. This fungus causes an anthracenose disease in northern joint vetch. It is specific to, and has a devastating effect on, its weed host. Optimum control of northern joint vetch was obtained when fresh spores of the fungus were applied at the rate of 187 billion spores/ ha in a volume of 94 I of water. Spores were produced in liquid culture and had to be used within 12 days after harvesting. This was a potential barrier to commercial use of the fungus because a spore preparation that could be stored and shipped without loss of activity was needed. The technology for drying and formulating spores was devel- oped by the Fermentation Division of the Upjohn Company which produces the com- mercial preparation Col1egoO. 161 Spore germination in the laboratory was 35 to 85070, and the shelf life of the formulation at 4C has been extended beyond 18 months. CollegoO is a dry powder composed of 15% spores and 85% inert ingredients. It is rehydrated before application. Improvement of spore germination has been achieved by rehydrating spores in small volumes of a 33% sugar solution before dilution in water.

    The spores were applied to rice and soybean fields with commercial application equipment at a rate of 94 I /ha containing 2 x lo6 viable spores per milliliter.159

    In another case, Alternaria macrospora has been used for the control of its suscep- tible host Anolla crislata,162 a weed causing significant economical damage in cotton fields. Inoculum was produced by growing the fungus in a submerged liquid culture. A preparation of air-dried conidia contained 1 x lo5 spores per gram and retained high viability by mixing the fungus with carriers such as vermiculite, clay particles, or corn- cob grits.

    Alternaria cassiae was also studied as a biocontrol agent of Cassia o b t ~ s i f o l i a . ~ ~ Phytophthora palmivora has been used to control milkweed vine, a major weed pest

    in citrus-growing areas of F10rida.l~~ The vine competes with the citrus trees for nu- trients and water and interferes with cultivation procedures. A Florida isolate of P . palmivora was found to be an efficient biocontrol agent. When 20 chlamydospores were applied per square centimeter of soil, over 90% of the vines died within 10 weeks. A preparation of this pathogen is produced commercially by Abbott Laboratories un- der the trade name DeVineO . DeVineB is available as a liquid suspension containing 6.7 x lo5 viable chlamydospores per milliliter. lS9

    Although the biological control of weeds has been studied extensively, 165.166 only two mycoherbicides have been registered. TeBeest and T e m p l e t ~ n ~ ~ described some ideas for future research. More work is required in the area of integrating biological control with classical control methods and in the development of pesticide-resistant strains. Protoplast fusion and recombinant DNA techniques may make it possible to combine the pathogenicity of two separate pathogens to form a more efficient mycoherbicide. More studies are needed about the safety of mycoherbicides in reference to their host range and in the toxicological aspects of mycoherbicide use.

    IV. CONCLUSIONS AND FUTURE PROSPECTS

    The material reviewed on beneficial associations between microorganisms and plants clearly shows that significant advances in our understanding of the physiology of their

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    interactions are necessary in order to improve these associations and convert the organ- isms involved into widely used commercial inoculants. This will be a painstaking proc- ess because of the complexity of the relationships among the introduced microorgan- ism, the microbial and physical environment on plant surfaces, the soil (an enormously complex environment in itself), and the plant.

    Research on the physiology of these interactions will need to be carried out at the most basic level by identification of molecules (proteins, polysaccharides, etc.) in- volved in the associations, by using modern biochemical methods of purification and analysis, and by the use of genetic and molecular genetic techniques to elucidate the mechanisms. An example is the use of transposon site mutations in cloned genes coding for one property (one protein) without affecting other properties related to prolifera- tion and survival of the introduced organisms during colonization of the plant sur- faces.

    There have been some significant advances at this level, but they have thus far had no practical impact. For example, advances have been made in the recognition of tri- folin-lectin of white clover and the saccharides of R. trifolii cell envelope," and the mapping, cloning, and expression of nod genes in Rhizobi~m'~ . '~ and nif genes in N2- fixing Other workers have shown that the T-DNA fraction of A. tumefa- ciens can serve as a vector for the introduction of foreign genes to plants.I6* In addi- tional research, a mutant lacking ice nucleation protein genes,129 plasmids of Pseudo- monads coding for pathogenicity, 169 and transposon-induced mutants of fluorescent pseudomonads lacking siderophores have been engineered. 139

    While studies of genetic properties and mapping and cloning genes involved in key processes related to the physiology of the interaction are underway, it will be feasible to select and use, by conventional methods, more efficient microbial strains affecting plants directly or indirectly as reviewed above.

    Probabilities of success in engineering microorganisms for inoculant production are estimated to be short to medium ( 5 to 10 years), where introduction of the desirable genes and their term expression in agriculturally important plants is a long-range goal (10 to 20 years).I7O An example is the introduction of the nifgene and its expression to a N-fixing plantI7' or the engineering of plants for resistance to a particular herbicide or Nevertheless, the rapidly developing area of molecular genetics and tech- nology may bring solutions to problems much sooner than estimated.

    predicted that no special ecological problems will be in- volved in the introduction of biological agents, plant, bacterium, or fungus containing foreign genes to the environment. Our experience with traditional practices, such as plant breeding and the use of microbial inoculants, has so far only benefited mankind.

    There is a need to improve the carriers and methods of delivery of microorganisms, to develop longer shelf life and survival, and to provide for optimal colonization rates of roots and leaves after inoculation. For example, applying bacteria suspended in granular preparations or embedded in resistant polysaccharide gels ensures survival of the inoculum and good colonization of roots in soils that may remain dry or cold for up to several weeks before root growth. All the delivery methods should be fully adapted to current agrotechnical procedures, thus minimizing delivery costs and ena- bling use in areas with less developed agriculture as well as in modern agriculture.

    For successful commercial production, the inoculants must work under a wide range of environments and climates. The farmers must be convinced that by their use yields are improved while expensive chemical treatments are avoided. It is expected that by the use of biological inoculants in agriculture the pollution of the environment by chemicals currently in use will be significantly diminished.

    In a recent article,

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