Application of Bacillus species in the control of root rot diseases of crop plants

Bio control potential of three Bacillus spp viz., Bacillus subtilis, B. thuringiensis and B. cereus, against soil borne root-infecting fungi on cowpea and mash bean plants were tested both in vitro and in vivo. All three species showed efficiency and produced nodules on mash bean and cow pea plants. In vitro dual culture plate method showed significant inhibition of Fusarium spp. by all these three species of Bacillus with the appearance of a prominent zone of inhibition while a maximum zone of inhibition of Fusarium spp. was observed by B. thuringiensis, whereas in case of Macrophomina phaseolina and Rhizoctonia solani, the highest zone of inhibition was observed by B. subtilis. Bacillus spp. used as seed dressing and soil drenching showed a significant increase in shoot length, shoot weight, root length and root weight in cow pea and mash bean plants. Maximum shoot length was observed in cow pea plants where Bacillus spp. were drenched in soil, whereas maximum root length and root weight in cow pea was observed when B. thuringiensis used as seed dressing. Seed dressing and soil drenching with species of Bacillus viz., B. subtilis, B. thuringiensis and B. cereus, were found to be an effective method for the control of soil borne root-infecting fungi like M. phaseolina, R. solani and Fusarium spp., on cow pea and mash bean plants.     

Biocontrol of chickpea root rot using endophytic actinobacteria

Eleven actinobacterial strains were isolated from different plants, lentil (Lens esculentus), chickpea (Cicer arietinum L.), pea (Pisum sativum), faba bean (Vicia faba) and wheat (Triticum vulgare) from Paskerville, South Australia. Isolates were characterized and identified morphologically as well as using 16S ribosomal RNA gene sequencing. Of the actinobacteria tested, 72% produced siderophores, 33% were positive for cyanogens production, and 11% showed phosphate solubility. All isolates had antimicrobial activity against Phytophthora medicaginis, Pythium irregulare and Botrytis cinerea. In a greenhouse experiment, actinobacteria with the highest biocontrol capabilities were tested for their ability to control Phytophthora root rot on chickpea. Both Streptomyces sp. BSA25 and WRA1 successfully suppressed Phytophthora root rot when coinoculated with either Mesorhizobium ciceri WSM1666 or Kaiuroo 3. Streptomyces sp. BSA25 with either rhizobial strain enhanced vegetative growth of root (7–11 fold) and shoot dry weights (2–3 fold) compared to infected control, whereas Streptomyces sp. WRA1 increased root and shoot dry weights by 8- and 4-fold, respectively when inoculated with M. ciceri WSM1666. We suggest that careful selection of actinobacteria should be considered when coinoculated with beneficial microorganisms as plant symbionts.     

Effects of salt on rhizobia and bradyrhizobia: a review

Rhizobia and bradyrhizobia strains vary in their tolerance to salt-stress. Rhizobium strains (fast-growers) are more salt-tolerant than strains of Bradyrhizobium (slow-growers). However, salt-tolerance in both genera is dependent upon ionic species, pH value, temperature, carbon source and the presence of osmoprotectant solutes. The harmful effect of salts on growth of both genera can be attributed to the specific ion effect rather than the osmotic effect. The salt-tolerance of different strains of rhizobia and bradyrhizobia is not related to their ecological origin. Data for salt tolerance of 684 strains of rhizobia and bradyrhizobia were collected from many reports. Most of the reports confound the effects of salt and express the concentrations of salts in percentage (%), electrical conductivity (dS m^-1), molar concentration (m ) or osmotic pressure (MPa) regardless of their differences. All the published data were compiled and recalculated from the different expressions to their equivalent molar concentration (m ) of NaCl. A suggested classification of salt-tolerance of rhizobia and bradyrhizobia from the compiled data is presented.     

Biocontrol of Rhizoctonia crown and root rot of sugar beet by binucleate Rhizoctonia spp. and Laetisaria arvalis

Two isolates of Laetisaria arvalis and 10 of binucleate Rhizoctonia spp. (BNR) from the Ohio sugar beet production area, were tested in the greenhouse and field for biocontrol of Rhizoctonia crown and root rot of sugar beet, caused by Rhizoctonia solani anastomosis group 2, type 2. L. arvalis was ineffective in standard greenhouse tests, and the single isolate used in the field was generally ineffective. Seven of 10 BNR isolates effectively controlled crown and root rot in greenhouse tests. Delayed application of biocontrol agents to plants 5 – 10 wk old was generally more effective than applications made at planting. A BNR isolate significantly reduced % plant loss and disease ratings and increased yield in a 1985 field test as compared with the control infested with R. solani alone. Two BNR isolates were effective in a 1986 field test and increased yields c. 22% in comparison to a L. arvalis treatment, which did not differ from the R. solani-infested control. The Ohio binucleate Rhizoctonia isolates appear to have considerable potential as applied biocontrol agents and may play a role in the natural ecology of R. solani in the sugar beet production area of Ohio.     

Population genetics of fungal diseases of plants

Although parasitism is one of the most common lifestyles among eukaryotes, population genetics on parasites lag for behind those on free-living organisms. Yet, the advent of molecular markers offers great tools for studying important processes, such as dispersal, mating systems, adaptation to host and speciation. Here we highlight some studies that used molecular markers to address questions about the population genetics of fungal (including oomycetes) plant pathogens. We conclude that population genetics approaches have provided tremendous insights into the biology of a few fungal parasites and warrant more wide use in phytopathology. However, theoretical advances are badly needed to best apply the existing methods. Fungi are of prime interest not only because they are major parasites of plants and animals, but they also constitute tractable and highly useful models for understanding evolutionary processes. We hope that the emerging field of fungal evolution will attract more evolutionary biologists in the near future.     

Cloning of nod gene regions from mesquite rhizobia and bradyrhizobia and nucleotide sequence of the nodD gene from mesquite rhizobia.

Nitrogen-fixing symbiosis between bacteria and the tree legume mesquite (Prosopis glandulosa) is important for the maintenance of many desert ecosystems. Genes essential for nodulation and for extending the host range to mesquite were isolated from cosmid libraries of Rhizobium (mesquite) sp. strain HW17b and Bradyrhizobium (mesquite) sp. strain HW10h and were shown to be closely linked. All of the cosmid clones of rhizobia that extended the host range of Rhizobium (Parasponia) sp. strain NGR234CS to mesquite also supported nodulation of a Sym- mesquite strain. The cosmid clones of bradyrhizobia that extended the host range of Rhizobium (Parasponia) sp. strain NGR234CS to mesquite were only able to confer nodulation ability in the Sym- mesquite strain if they also contained a nodD-hybridizing region. Subclones containing just the nodD genes of either genus did not extend the host range of Rhizobium (Parasponia) sp. to mesquite, indicating that the nodD gene is insufficient for mesquite nodulation. The nodD gene region is conserved among mesquite-nodulating rhizobia regardless of the soil depth from which they were collected, indicating descent from a common ancestor. In a tree of distance relationships, the NodD amino acid sequence from mesquite rhizobia clusters with homologs from symbionts that can infect both herbaceous and tree legumes, including Rhizobium tropici, Rhizobium leguminosarum bv; phaseoli, Rhizobium loti, and Bradyrhizobium japonicum.     

Biological control of the root rot and wilt diseases ofLens culinaris medic

Summary Biological control visualizes a control of disease with the help of living organisms. In case of root disease, organisms occur in soil which suppress the activity of disease fungi. In the present investigation antagonists have been isolated and used in the control of root rot and wilt disease ofLens culinaris caused byS. rolfsii andF. oxysporum respectively.Isolation of antagonistic micro-organisms were done from the rhizosphere soil of Bombay 20 and local varieties and from field soil. Antagonists were screened against certain test organisms and pathogens. The selected antagonists were used for the control of the diseases of lentil crop by inoculating the antagonists in the soil and thereby to see the effect of decreased plant disease in sterilized and unsterilized soil.Amendments in the form of different organic materials were also given to control the diseases in natural soil.The most effective results were obtained withT. viride, Streptomyces gougeroti Streptomyces species 12 and 13, bacterial isolates no. 16 and 18 in case ofF. oxysporum f.lentis inoculated pots, where there was no disease. In case ofS. rolfsii inoculated pots not a single antagonist was able to eradicate the disease completely.Portion of the Ph. D. thesis accepted by the University of Saugor, Sagar, India.     

Effects of crop residues in soil on phymatotrichum root rot of cotton

The effects of cropping pea or barley and the decomposition of their residues on Phymatotrichum root rot of cotton were studied for five years. Little or no changes occurred in pH, content of carbon or moisture in the soil, but there were significant increases in populations of competitive microflora. Best control was obtained in infested soil which was cropped to either pea or barley in winter or in soil which received the higher tonnage of Papago pea plants. Reduction in incidence of root rot may be attributed to premature germination of overwintering propagules making hyphae vulnerable to the chemicals of decomposition and to competition of microflora in the soil.Former Graduate Assistant in Research, Associate Professor, Professor, and Former Professor of Plant Pathology     

The binding of zinc in root cells of crop plants by phytic acid

Appreciable quantities of Zn are bound as Zn phytate (myo-inositol kis-hexaphosphate) within small vacuoles of cortical cells in the elongation zone of root tips of zinc tolerant Deschampsia caespitosa. These Zn/P-containing globular deposits have now been shown to occur in the roots of soybean, lucerne, lupins, tomato, rapeseed, cabbage, radish, wheat and maize. The globules are most frequent in the endodermis and pericycle but may also occur in the stele and inner cortex. The X-ray data again confirmed the presence of phytate with a relatively stable proportion of Zn and a species-dependent, variable, proportion of K, Mg and Ca to P.Analysis of soybean plants by atomic absorption spectroscopy showed that the Zn concentration in the shoots doubled in response to an increase in Zn supply from 1 to 100 M while the concentration of Zn in the root symplast was approximately 22 times greater than in the shoot, suggesting restricted transport to the shoot. It is suggested that the genetic expression of the capacity to bind heavy metals by means of phytate in endodermal cells may provide a strategy for keeping the above-ground content of heavy metals low. It may be possible to incorporate the trait into transformed roots that can be utilized for the treatment of industrial wastes.     

Biocontrol activity of salt tolerant Streptomyces isolates against phytopathogens causing root rot of sugar beet

Biological control of fungi causing root rot on sugar beet by native Streptomyces isolates (C and S2) was evaluated in this study. The dry weight and colony forming unit (CFU) of S2 and C increased when 300 mM NaCl was added to medium. The in vitro antagonism assays showed that both isolates had inhibitory effect against Rhizoctonia solani AG-2, Fusarium solani and Phytophthora drechsleri. In dual culture, Streptomyces isolate C inhibited mycelial growth of R. solani, F. solani and P. drechsleri 45%, 53% and 26%, respectively. NaCl treatment of medium increased biocontrol activity of soluble and volatile compounds of isolate C and S2. After salt treatment, growth inhibition of R. solani, F. solani and P. drechsleri by isolate C increased up to 59%, 70% and 79%, respectively. To elucidate the mode of antagonism, protease, chitinase, beta glucanase, cellulase, lipase and α-amylase activity and siderophore and salicylic acid (SA) production were evaluated. Both isolates showed protease, chitinase and α-amylase activity. Also, biosynthesis of siderophore was detectable for both isolates. Production of siderophore and activity of protease and α-amylase increased after adding salt for both isolates. In contrast, chitinase activity decreased significantly. Production of SA, beta glucanase and lipase by isolate S2 and biosynthesis of cellulase by isolate C were observed in presence and absence of NaCl. Soil treatment with Streptomyces isolate C inhibited root rot of sugar beet caused by P. drechsleri, R. solani and F. solani. Results of this study showed that these two Streptomyces isolates had potential to be utilized as biocontrol agent against fungal diseases especially in saline soils.     

Biocontrol of Phytophthora cactorum the causal agent of root and crown rot on apple (Malus domestica) by formulated Pseudomonas fluorescens

In this study 284 isolates were isolated of apple trees' rhizosphere from Iran and 128 isolates were obtained from the collection of Research Department of Biological Control of University of Tehran. Four strains (P60, P61, P96, and P97) of Pseudomonas fluorescens were selected for greenhouse trials. The results of greenhouse trials showed dipping the crown and root of apple seedlings (MM106) combined with soil drench was more effective than dipping the crown and root on reducing the disease. After 6 weeks, strain P60 in dipping method combined with soil drench with 70% control, exhibited greatest effect on reducing the crown and root rot and was more effective than the fungicide metalaxyl-mancozeb. After 12 weeks, strains P60 and P96 in dipping method combined with soil drench with 55.6% and 44.5% control respectively, exhibited greatest effects on reducing the diseases Study of media on growth rate populations of effective strains exhibited that the beet molasses yeast extract (1:1) had more effect than nutrient broth(NB) medium. The initial high populations of powder formulations of strains P60 and P96 decreased during the storage at 4 and 25 degrees C over a 150-day period. In addition, formulations of strains stored at 4 degrees C had longer shelf life than those stored at 25 degrees C. In glasshouse trials, after 6 weeks, formulation of strain P60 and unformulated P60, obtained from NB medium and formulated P60, obtained from molasses yeast extract medium, and metalaxyl-mancozeb had highest effect on reducing the disease on apple rootstocks. After 12 weeks, formulation of strain P60 and unformulated bacteria obtained from both media, and metalaxyl-mancozeb with 57.1% control showed greatest effect on reducing the disease.     

Wheat root colonization and nitrogenase activity by Azospirillum isolates from crop plants in Korea

Nitrogen-fixing bacteria were isolated from the rhizosphere of different crops of Korea. A total of 16 isolates were selected and characterized. Thirteen of the isolates produced characteristics similar to those of the reference strains of Azospirillum, and the remaining 3 isolates were found to be Enterobacter spp. The isolates could be categorized into 3 groups based on their ARDRA patterns, and the first 2 groups comprised Azospirillum brasilense and Azospirillum lipoferum. The acetylene reduction activity (ARA) of these isolates was determined for free cultures and in association with wheat roots. There was no correlation between pure culture and plant-associated nitrogenase activity of the different strains. The isolates that showed higher nitrogenase activities in association with wheat roots in each group were selected and sequenced. Isolates of Azospirillum brasilense CW301, Azospirillum brasilense CW903, and Azospirillum lipoferum CW1503 were selected to study colonization in association with wheat roots. We observed higher expression of beta-galactosidase activity in A. brasilense strains than in A. lipoferum strains, which could be attributed to their higher population in association with wheat roots. All strains tested colonized and exhibited the strongest beta-galactosidase activity at the sites of lateral roots emergence.     

Characterization of root hair cell walls as potential barriers to the infection of plants by rhizobia : the carbohydrate component

The sugar compositions of root hairs of a variety of plant species were determined. Root hairs of legumes had very similar compositions, whereas those of different families varied widely. Only approximately 50% of the weight of the root hairs of legumes could be accounted for by sugar. Up to 80% of the weight of root hairs from other sources could be accounted for by sugars. Protein made up 5 to 8% of the weight of root hairs of dicots but only 1.3% in corn. Comparison between cell walls from various root cell types within legumes showed that the polysaccharide compositions of root epidermal, root hair, and root cortical cells were very similar. Cotton root hairs were markedly different from walls of mesophyll and epidermal cells of cotyledons from cotton.     

Biocontrol of avocado dematophora root rot by antagonistic Pseudomonas fluorescens PCL1606 correlates with the production of 2-hexyl 5-propyl resorcinol

A collection of 905 bacterial isolates from the rhizospheres of healthy avocado trees was obtained and screened for antagonistic activity against Dematophora necatrix, the cause of avocado Dematophora root rot (also called white root rot). A set of eight strains was selected on the basis of growth inhibitory activity against D. necatrix and several other important soilborne phytopathogenic fungi. After typing of these strains, they were classified as belonging to Pseudomonas chlororaphis, Pseudomonas fluorescens, and Pseudomonas putida. The eight antagonistic Pseudomonas spp. were analyzed for their secretion of hydrogen cyanide, hydrolytic enzymes, and antifungal metabolites. P. chlororaphis strains produced the antibiotic phenazine-1-carboxylic acid and phenazine-1-carboxamide. Upon testing the biocontrol ability of these strains in a newly developed avocado-D. necatrix test system and in a tomato-F oxysporum test system, it became apparent that P. fluorescens PCL1606 exhibited the highest biocontrol ability. The major antifungal activity produced by strain P. fluorescens PCL1606 did not correspond to any of the major classes of antifungal antibiotics produced by Pseudomonas biocontrol strains. This compound was purified and subsequently identified as 2-hexyl 5-propyl resorcinol (HPR). To study the role of HPR in biocontrol activity, two Tn5 mutants of P. fluorescens PCL1606 impaired in antagonistic activity were selected. These mutants were shown to impair HRP production and showed a decrease in biocontrol activity. As far as we know, this is the first report of a Pseudomonas biocontrol strain that produces HPR in which the production of this compound correlates with its biocontrol activity.     

Engineering nitrogen use efficient crop plants: the current status

In the last 40 years the amount of synthetic nitrogen (N) applied to crops has risen drastically, resulting in significant increases in yield but with considerable impacts on the environment. A requirement for crops that require decreased N fertilizer levels has been recognized in the call for a ‘Second Green Revolution’ and research in the field of nitrogen use efficiency (NUE) has continued to grow. This has prompted a search to identify genes that improve the NUE of crop plants, with candidate NUE genes existing in pathways relating to N uptake, assimilation, amino acid biosynthesis, C/N storage and metabolism, signalling and regulation of N metabolism and translocation, remobilization and senescence. Herein is a review of the approaches taken to determine possible NUE candidate genes, an overview of experimental study of these genes as effectors of NUE in both cereal and non‐cereal plants and the processes of commercialization of enhanced NUE crop plants. Patents issued regarding increased NUE in plants as well as gene pyramiding studies are also discussed as well as future directions of NUE research.     

Biocontrol bacteria selected by a direct plant protection strategy against avocado white root rot show antagonism as a prevalent trait

Aim: This study was undertaken to study bacterial strains obtained directly for their efficient direct control of the avocado white root rot, thus avoiding prescreening by any other possible mechanism of biocontrol which could bias the selection. Methods and Results: A collection of 330 bacterial isolates was obtained from the roots and soil of healthy avocado trees. One hundred and forty‐three representative bacterial isolates were tested in an avocado/Rosellinia test system, resulting in 22 presumptive protective strains, all of them identified mainly as Pseudomonas and Bacillus species. These 22 candidate strains were screened in a more accurate biocontrol trial, confirming protection of some strains (4 out of the 22). Analyses of the potential bacterial traits involved in the biocontrol activity suggest that different traits could act jointly in the final biocontrol response, but any of these traits were neither sufficient nor generalized for all the active bacteria. All the protective strains selected were antagonistic against some fungal root pathogens. Conclusions: Diverse bacteria with biocontrol activity could be obtained by a direct plant protection strategy of selection. All the biocontrol strains finally selected in this work were antagonistic, showing that antagonism is a prevalent trait in the biocontrol bacteria selected by a direct plant protection strategy. Significance and Impact of the Study: This is the first report on the isolation of biocontrol bacterial strains using direct plant protection strategy in the system avocado/Rosellinia. Characterization of selected biocontrol bacterial strains obtained by a direct plant protection strategy showed that antagonism is a prevalent trait in the selected strains in this experimental system. This suggests that antagonism could be used as useful strategy to select biocontrol strains.     

Green manures and crop sequences influence alfalfa root rot and pathogen inhibitory activity among soil-borne streptomycetes

A two-year trial was conducted to determine the effects of green manures and crop sequences on plant disease, streptomycete and bacterial densities, and inhibitory activity of indigenous streptomycetes against four target pathogens. Green manure treatments, buckwheat (Fagopyrum esculentum L.), canola (Brassica napus L.), sorghum-sudangrass (Sorghum bicolor) (L.) Moench × Sorghum sudanense (Piper) Stapf.), and fallow control were tested in conjunction with three crop sequences in a Phytophthora-infested soil placed in containers. Alfalfa (Medicago sativa L.), potato (Solanum tubersoum L.), or corn (Zea mays L.) was grown in the first year, and alfalfa was grown in all containers in the second year. Compared to fallow controls, alfalfa grown in sorghum-sudangrass- or buckwheat-treated soil had significantly greater stand counts and total biomass, respectively. In addition, alfalfa grown in fallow-treated soils had the greatest Phytophthora root rot as a function of stand count. Crop rotation also had a significant effect on alfalfa root rot and yield. Potato scab disease intensity was greatest on tubers grown in fallow-treated soils, while tubers grown in canola-treated soils had the highest yields (total tuber weight). Green-manure-treated soils tended to have greater streptomycete and bacterial densities than fallow-treated soils. In addition, buckwheat- or sorghum-sudangrass-treated soils had greater proportions of streptomycetes that were antagonistic against the target pathogens than fallow-treated soils. The proportion of antagonists in soil was negatively correlated with alfalfa root rot, and positively correlated with alfalfa stand counts. Inhibitory activity of the streptomycetes was also negatively correlated with potato scab and positively correlated with potato yield. These data suggest that green manures may provide a strategy for increasing pathogen inhibitory activity within the streptomycete community in soil, and, in conjunction with crop rotation, may contribute to the control of a diverse collection of soil-borne plant pathogens on multiple crop species.     

Genetic engineering of crop plants for fungal resistance: role of antifungal genes

Fungal diseases damage crop plants and affect agricultural production. Transgenic plants have been produced by inserting antifungal genes to confer resistance against fungal pathogens. Genes of fungal cell wall-degrading enzymes, such as chitinase and glucanase, are frequently used to produce fungal-resistant transgenic crop plants. In this review, we summarize the details of various transformation studies to develop fungal resistance in crop plants.