Microbial Inoculants as Crop-Yield Enhancers

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<ul><li><p>Volume 6, Issue 1 (1987) 61 </p><p>MICROBIAL INOCULANTS AS CROP-YIELD ENHANCERS </p><p>Authors: Yaacov Okon Yitzhak Hadar Department of Plant Pathology and Microbiology </p><p>Faculty of Agriculture The Hebrew University of Jerusalem Rehovot, Israel </p><p>Referee: Ralph Baker Department of Plant Pathology and Weed Science Colorado State University Fort Collins. Colorado </p><p>I. INTRODUCTION </p><p>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. </p><p>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. </p><p>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. </p><p>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. </p><p>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. </p><p>Cri</p><p>tical</p><p> Rev</p><p>iew</p><p>s in</p><p> Bio</p><p>tech</p><p>nolo</p><p>gy D</p><p>ownl</p><p>oade</p><p>d fr</p><p>om in</p><p>form</p><p>ahea</p><p>lthca</p><p>re.c</p><p>om b</p><p>y Y</p><p>ork </p><p>Uni</p><p>vers</p><p>ity L</p><p>ibra</p><p>ries</p><p> on </p><p>08/1</p><p>3/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p> onl</p><p>y.</p></li><li><p>62 CRC Critical Reviews in Biotechnology </p><p>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. </p><p>11. DIRECTLY BENEFICIAL INOCULANTS </p><p>A. Rhizobium Legume Symbioses The relationship between Rhizobium and legumes is one of the most extensively </p><p>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 . ~ - ~ </p><p>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. </p><p>1. Taxonomy The division of Rhizobium into six species according to "cross inoculation" groups, </p><p>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). </p><p>2. The Infection Process and Function of the Nodule Rhizobia vary in their ability to survive in the soil, but proliferation takes place </p><p>mainly in the rhizosphere of host and nonhost plant^.'^^^^ In some soil types, highly </p><p>Cri</p><p>tical</p><p> Rev</p><p>iew</p><p>s in</p><p> Bio</p><p>tech</p><p>nolo</p><p>gy D</p><p>ownl</p><p>oade</p><p>d fr</p><p>om in</p><p>form</p><p>ahea</p><p>lthca</p><p>re.c</p><p>om b</p><p>y Y</p><p>ork </p><p>Uni</p><p>vers</p><p>ity L</p><p>ibra</p><p>ries</p><p> on </p><p>08/1</p><p>3/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p> onl</p><p>y.</p></li><li><p>Volume 6, Issue 1 (1987) 63 </p><p>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. </p><p>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. </p><p>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 </p><p>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. </p><p>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 </p><p>Cri</p><p>tical</p><p> Rev</p><p>iew</p><p>s in</p><p> Bio</p><p>tech</p><p>nolo</p><p>gy D</p><p>ownl</p><p>oade</p><p>d fr</p><p>om in</p><p>form</p><p>ahea</p><p>lthca</p><p>re.c</p><p>om b</p><p>y Y</p><p>ork </p><p>Uni</p><p>vers</p><p>ity L</p><p>ibra</p><p>ries</p><p> on </p><p>08/1</p><p>3/14</p><p>For </p><p>pers</p><p>onal</p><p> use</p><p> onl</p><p>y.</p></li><li><p>64 CRC Critical Reviews in Biotechnology </p><p>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 </p><p>3. Rhizobium Znoculants and Inoculation Technologies a. Rhizobium Strain Selection </p><p>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. </p><p>1. </p><p>2. </p><p>3. </p><p>4. </p><p>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...</p></li></ul>