Search

Production of perithecia, asci, and ascospores by Indian isolates of Magnaporthe grisea is rare and has not been found among the Southern Indian isolates of the blast pathogen. From among 138 monoconidial isolates that infect rice and other hosts, we now report the distribution of mating types (MAT1-1 and MAT1-2) of M. grisea in finger millet and paragrass (Brachiaria mutica)-infecting isolates. Twenty-eight of the 96 finger millet isolates, 5 of the 16 paragrass isolates, and none of the 26 rice isolates produced fertile perithecia in laboratory matings with fertile testers. Backcrosses of ascospore progenies to the parental M. grisea isolate but not to the tester strain resulted in fertile perithecial formation, and a further backcrossing scheme indicated definite fertility patterns of Mendelian inheritance in M. grisea.

About 200 isolates of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae were obtained from infected rice (Oryza sativa L.) leaf samples from the Palghat District of Kerala State in southern India during January and February, 1998, when there was a severe epidemic of bacterial blight in the farmers' fields. One hundred and forty of these isolates were assayed for pathogenicity on rice cv. IR24 (no R gene), IRBB21 (Xa21), and NH56 (Xa4+xa5+xa13+Xa21) at their maximum tillering stage by the standard clip-inoculation technique (1) at the Regional Agricultural Research Station (Kerala Agricultural University), Pattambi. In two separate experiments, all the isolates were found to be pathogenic to IR24, producing lesions greater than 10 cm in length. None of the isolates induced susceptible symptoms on cv. NH56 and the average lesion length was less than 2 cm. Twenty of the 140 isolates, however, showed virulence to IRBB21 (seeds supplied by S. R. McCouch, Cornell University, and by P. Ronald, University of California-Davis) and induced lesions of 10.0 to 18.0 cm. We report here the presence of Indian races of X. oryzae pv. oryzae that are virulent to IRBB21 (Xa21), contradicting an earlier report of avirulence (2). These results suggest that the deployment of Xa21 alone will not be a sound strategy for the management of bacterial blight in southern India. References: (1) S. S. Gnanamanickam et al. Plant Dis. 78:173, 1994. (2) G.-L.Wang et al. Mol. Plant-Microbe Interact. 9:850, 1996.

Use of BTH to evaluate the disease severity and induction of systemic resistance in rice to bacterial blight caused by Xanthomonas oryzae pv. oryzae is investigated. A new batch of 25 isolates of Xanthomonas oryzae pv. oryzae was obtained from infected rice lead tissues collected from Pattambi, Kerala, south India. Their identification was confirmed by the plant inoculation test on to IR24 rice plants which produced characteristic bacterial blight lesions. Among the 25 of X.o. pv. oryzae, four of the isolates were also virulent to IRBB21 rice plants (a near isogenic line of IR24) which carry the Xa-21 gene for BB resistance. The results confirm that there are pathogen strains in India which can overcome Xa-21. Development of BB lesions developed in IR24 (BB susceptible) plants after they were treated with BTH applications either as seed treatment or as foliar spray at 0.1, 0.5, 0.1 and 2.0 mM concentrations showed that even at 2.0 mM concentrations, IR24 plants were still susceptible to the pathogen. Ther...

Efficient strains of bacterial antagonists for Magnaporthe grisea were identified through laboratory dual-plate assays. Two bacterial strains of Pseudomonas fluorescens and two strains of Bacillus were tested against 30 isolates of M. grisea that cause Setaria blast for their inhibition of the fungus. On plates spread with the fungus mycelium, 12 of the isolates had a zone of inhibition caused by the bacteria that ranged between 2.5 and 3.0 cm in diameter. In the field trails, the bacterial strains Pf-52 and KRU-22 suppressed disease by 86.64% and 87.87% respectively, in Setaria italica line 1, and 86.64% and 83.21% in line 2. The mixture of all four strains afforded 88.87% (line 1) and 88.80% (line 2) blast suppression and this was the highest level of biological blast control observed. In addition to disease suppression, the bacterial strains significantly increased root and shoot length when compared with untreated plants. Of the four strains, Pf-52 , KRU-22 and the mixture of all four strains showed significant increases of root length of 10.02, 7.44 and 13.29 cm (line 1), and 9.2, 5.7 and 13.11 cm (line 2) in the root growth stage. As in the case of root length, Pf-52 , KRU-22 and the mixture of all four strains showed significant increases of shoot length of 11.38, 10.42 and 11.99 cm (line 1), and 9.87, 11.44 and 12.11 cm (line 2) in the shoot growth stage.

Jatropha (Jatropha curcas L.) is a non-edible oil- seed plant with adaptability to marginal semi-arid lands and wastelands. The Indian Government is promoting jatropha to reduce dependence on the crude oil and to achieve energy independence by the year 2012, under the National Biodiesel Mission. Selected strains of Bacillus spp., either supplemented with or without chitin, were tested for their ability to promote growth of jatropha seedlings in pot culture studies. The strains supported growth of jatropha seedlings up to 42 days after sowing. Among all strains, Bacillus pumilus (IM-3) supplemented with chitin showed over all plant growth promotion effect resulting in enhanced shoot length (113%), dry shoot mass (360%), dry root mass (467%), dry total plant mass (346%), leaf area (256%), and chlorophyll content (74%) over control. Treating seeds with strain IM-3 without chitin resulted in enhanced dry shoot mass (473%), dry total plant mass (407%), and chlorophyll content (82%). However, Bacillus polymyxa (KRU-22) with chitin supported maximum root length (143%). Either strain IM-3 alone or in combination with other promising strains could be promoted further for enhanced initial seedling growth of jatropha.

A high level of genetic polymorphism was detected among Indian isolates of Xanthomonas oryzae pv. oryzae using hypervariable probes such as a microsatellite oligonucleotide, probe (TG)10, a human minisatellite probe, pV47, an avirulence gene probe, avrXa10 and a repeat clone, pBS101. These DNA probes detected multiple loci in the bacterial genome generating complex DNA fingerprints and differentiated all of the bacterial isolates. Analysis of fingerprints indicated that pV47, (TG)10 and pBS101 have a lower probability of identical match than avrXa10 and therefore are potential probes for DNA fingerprinting and variability analysis of Xanthomonas oryzae pv. oryzae pathogen populations. Cluster analysis based on hybridization patterns using all of the above probes showed five groups at 56% similarity. Studies on the methylation patterns of isolates representing the three important races of X. oryzae pv. oryzae indicated more methylation in the most virulent isolate, suggesting a possible role of methylation in pathogenicity.

Bacterial antagonists of both fluorescent and nonfluorescent groups were screened for in-vitro inhibition of the rice sheath blight (ShB) fungus Rhizoctonai solani Kuhn and chitinase production in the laboratory. Twelve percent of the total 1,757 strains screened inhibited R. solani while 31% of the total 1,366 strains tested were positive for chitinase activity. The efficient strains were then evaluated in the field for ShB suppression. Two strains from each of the three seed-bed experiments were chosen for the field test in a hot-spot location. Additional treatments were a Bacillus and validamycin, the fungicide. There was no correlation between chitinase activity in the antagonists and ShB suppression in the seed-bed or field plots. Two most efficient Pseudomonas putida and P. fluorescens strains afforded 68 and 52% ShB suppression while validamycin afforded 27% disease control.

Fluorescent and nonfluorescent strains of bacteria isolated from rice rhizospheres on the International Rice Research Institute (IRRI) farm were evaluated for in vitro antibiosis towards the sheath blight (ShB) pathogen Rhizoctonia solani, and for suppression of ShB in detached rice leaves. Efficient strains were located on the basis of consistent performance in two laboratory tests. Among nine efficient strains, 3 strains were identified as Pseudomonas fluorescens, 5 strains were tentatively identified as Bacillus spp. and one strain was identified as Enterobacter. In three greenhouse tests lowland rice soils with optimum pH for rice growth (pH 5.5–6.5), acidic pH (pH 5.0) and boron toxicity were found more suitable for biological control of ShB and, less frequently, also yield increases than were alkaline (pH 6.9) and zinc-deficient soils. Bacterial treatments afforded significant ShB reductions in 3 field experiments, but no significant yield increases resulted. In direct-seeded rice best performances by bacterial treatments in terms of ShB suppression were 66 and 98% during DS 1988 and WS 1988, respectively, which were comparable to or better than the performance of validamycin (a fungicide routinely used for ShB control) which afforded 42 and 63% disease suppression, respectively, in the same experiments. Although bacterial treatments caused ShB reductions both in direct-seeded and transplanted rice crops, disease control was more pronounced in direct-seeded than in transplanted crops. These results indicate that carefully selected strains of bacterial antagonists have the potential for ShB suppression in rice at least in areas where direct-seeding is practised.

In laboratory tests Sarocladium oryzae, the sheath rot pathogen of rice was found to inhibit the mycelial growth of other stem-attacking rice pathogens. Among those inhibited, Sclerotium oryzae and Gaeumannomyces graminis var. graminis were most sensitive while Pyricularia oryzae and Rhizoctonia solani were less sensitive. Tissue-based tests made with rice culm segments established that Sarocladium oryzae inhibits mycelial growth and delays sclerotium formation in R. solani. Cerulenin, the toxin produced by Sarocladium oryzae showed a toxicity pattern towards rice pathogens similar to that of Sarocladium oryzae. The stem rot pathogen, Sclerotium oryzae was most sensitive to cerulenin. In two greenhouse experiments, IR58 rice plants inoculated with Sarocladium oryzae alone or together with Sclerotium oryzae, G. graminis var. graminis or R. solani were found to have reduced plant height and increased tiller number. Sheath rot severity increased when Sarocladium oryzae was inoculated as a single pathogen or together with others. Sheath rot inoculation reduced stem rot in rice plants by 76 and 58%, respectively, in Experiment 1 and 2. By its known antagonistic interaction towards stem rot and crown sheath rot pathogens which are sensitive to it and by other unknown interactions, sheath rot emerges as the dominant disease.

From among 125 strains of fluorescent and 52 strains of nonfluorescent bacteria initially screened in the laboratory for their antibiosis towards the bacterial wilt pathogen, Pseudomonas solanacearum, strain Pfcp of Pseudomonas fluorescens and strains B33 and B36 of Bacillus spp., were chosen and evaluated further in greenhouse and field tests. Pfcp treated banana (Musa balbisiana), eggplant and tomato plants were protected from wilt upto 50, 61 and 95% in greenhouse and upto 50, 49 and 36% respectively in field. Protection afforded by the Bacillus strains was lower. In bacteria-treated plants which were subsequently inoculated with P. solanacearum plant height and biomass values increased and were close to those of nontreated and noninoculated control plants.

Two strains of rice-associated pseudomonads Pseudomonas fluorescens 7-14 and P. putida V14i caused an induced systemic resistance (ISR) in three cultivars of rice. ISR response coincided with increased levels of salicylic acid and biological suppression (25%) of rice blast disease. When the strains of the bacteria with lacZY molecular marker tracking system were applied to rice seeds as a seed-coat, they persisted on rice roots for entire duration of the crop (110 days) but migrated to the rice shoot only up to 14 to 16 days. Bacteria applied as a root-dip did not migrate to aerial parts of the rice plant. Bacteria infiltrated into the rice stem survived for 20 days after the treatment and were not detected on the surface of the stem. It is suggested that the ISR triggered by these bacteria which were spatially separated from the rice blast pathogen, is an important mechanism for the biological suppression of rice blast.

Rice is the world’s most important and staple food for more than a third of the world’s population. Understandably, then there is a consistent demand for higher yields of rice grains through breeding and genetic manipulations, judicious use of fertilizers and water, as well as control of insect pests and diseases.

We have identified 25 MGR-fingerprint lineages of the rice blast fungus in southern India in samplings from 1991–95; some lineages appear to be geographically limited to certain states. In artificial inoculation assays, each lineage has a definable virulence spectrum on rice cultivars that is limited by avirulence shared by all lineage members. We are testing a lineage-exclusion resistance breeding strategy, i.e., combining resistances that, in complement, exclude all lineages in the pathogen population, in contrast to the conventional strategy that combines resistances to exclude detected pathotypes. In two-semester trials in a blast nursery at Pattambi, Kerala we evaluated field performance of rice lines constructed to pyramid two dominant blast resistance genes from the near-isogenic lines C101A51 (bearing Pi-2(t)) and C101LAC (bearing Pi-1(t)); previous inoculation assays indicated that no lineage in Kerala or adjoining states was compatible with Pi-2(t) and that Pi-1(t) was effective against most lineages. All pyramids remained uninfected during trials while many cultivars, including C101LAC but not C101A51, were heavily infected. Our pathogenicity survey results indicate, however, that the durability of these pyramids is better tested in other states (Tamil Nadu, Maharastra and Orissa), where we have detected isolates differentially compatible with both resistance genes. Additional pyramids of C101A51 and C103TTP (allegedly bearing a version of the Pi-1 (t) gene) were constructed. Using isolates from both India and Colombia, we demonstrated that the gene from C103TTP (renamed as Pilb(t)) was expressed as a recessive phenotype. We are currently working on introgressing commercial cultivars used in various regions of India with available lineage-excluding resistances.

By MGR-DNA fingerprinting analyses, we identified 29 lineages of Magnaporthe grisea among rice blast pathogen population in Southern India. The population structure of M. grisea in different states of India was also characterized by a Pot2-based rep-PCR method. The latter method identified 1 to 22 bands in the fingerprints of rice-infecting and less than 10 bands in the fingerprints of non rice-infecting strains of M. grisea which were in the size range of 0.4 to 23 kb. Lineage-exclusion assays showed that a combination of Pi-1 and Pi-2 genes for blast-resistance can exclude all the lineages identified. This virulence characteristic of M grisea remained stable while its lineage-composition showed drastic changes over the period 1993–97 in rice-growing areas of Kerala where annual blast epidemics is common. Therefore, gene pyramids of Pi-1/Pi-1b+Pi-2 were constructed in the CO39 (blast-susceptible) genomic background and such pyramids were resistant to blast in hot-spot locations. The CO39 pyramid now serves as the R-donor for introgressing the Pi-1+Pi-2 genes for blast resistance into cultivars such as Jyothi and IR50 which are high-yielding but blast-prone to make them durably resistant.

As part of efforts to develop alternate strategies for rice disease management, more than 400 strains of bacteria isolated from IRRI rice fields were screened in the laboratory for antagonism towards Pyricularia oryzae and Rhizoctonia solani, the fungal pathogens of blast and sheath blight respectively. On the basis of growth-inhibition in plate assays, 9 bacterial strains which included 3 strains of Pseudomonas fluorescens, 5 strains of Bacillus spp and a strain of Enterobacter were identified. Diameter of inhibition zones ranged from 20–40 mm against P. oryzae and from 20–31 mm against R. solani. These bacterial strains also suppressed sheath blight (Sh-B) in detached rice leaves. They were further evaluated in field tests for suppression of blast and Sh-B when applied as seed treatments or as seed treatments plus sprays. In three field experiments conducted in the IRRI farm, bacterial treatments afforded significant Sh-B control and performed better than validamycin, the fungicide routinely used for Sh-B control. Suppression of Sh-B was more pronounced in direct-seeded rice than in tranplanted rice. Bacterial treatments reduced leaf blast severities by 50–73% in var-IR50 and 34–80% in var C-22 in lowland experiment and 47–57% reduction in var. UPLRi-5 in an upland experiment. Further investigations with a Pseudomonas strain, 7–14 have shown that it produces 2 fluorescent antiblast antibiotics (AB1 and AB2) in culture which at 1.0 ppm inhibit conidial germination of P. oryzae and afford 90–92% reduction in leaf blast development in treated rice (var. IR 50) seedlings. Therefore, biological control of blast by Pseudomonas is antibiotic-mediated.

Four semiselective media that varied with respect to carbon source, amino acids, and antibiotics were evaluated for the growth of various strains of the rice bacterial blight pathogen, Xanthomonas oryzae pv. oryzae in pure and mixed culture. Plating efficiencies in pure culture ranged from 40 to 90% on these media, and colonies developed in 3-7 days, depending on the strain of X. o. oryzae. In mixtures with Erwinia herbicola and Pseudomonas putida(1:1:1), X. o. oryzae was recovered only on XOS medium. Recovery of a faster growing X. o. oryzae strain (X1-5) was improved by adding 50-100 mg/L of FeEDTA to XOS medium, but recovery of other strains was reduced or unaffected

Native strains ofPseudomonas fluorescens exhibitedin vitro antibiosis towards isolates of races 1 and 4 ofFusarium oxysporum f.sp.cubense, the Panama wilt pathogen of banana. The seedlings ofMusa balbisiana seedlings treated withP. fluorescens showed less severe wilting and internal discolouration due toF. oxysporum f.sp.cubense infection in greenhouse experiments. In addition to suppressing Panama wilt, bacterized seedlings ofM. balbisiana also showed better root growth and enhanced plant height.

Coronatine was detected in culture filtrates of 12 out of 19 pathogenic strains of Pseudomonas glycinea examined, but not in culture filtrates of strains of P. phaseolicola, P. syringae, or P. tabaci. A reversed-phase high-performance liquid chromatographic procedure was developed for coronatine quantitation. Generally, coronatine production by strains in culture correlated with their ability to induce systemic symptoms (chlorosis and stunting) in inoculated soybean plants. Application of purified preparations of coronatine to unifoliate leaves of soybean plants resulted in localized chlorosis, development of chlorosis in subsequently developing trifoliate leaves, and stunting of plant growth, similar to symptoms induced by infection. Coronatine was demonstrated in soybean leaves infected with P. glycinea but was not detected in healthy leaves. The results indicate that coronatine can play an important role in the development of symptoms of bacterial blight of soybean, but the demonstration that some pathogenic strains do not produce coronatine indicates that it may not be essential for pathogenicity.

Pseudomonas fluorescens strains antagonistic to Sarocladium oryzae , the sheath rot (Sh-R) pathogen of rice ( Oryza sativa L.), were evaluated in greenhouse and field tests for suppression of Sh-R severity and enhancement of grain yields of rice. Imprints of rice seedlings and a direct-observation technique of staining roots with fluorochromes confirmed the association of P. fluorescens with roots and the ability of the strain to move along shoot tips. In greenhouse tests, P. fluorescens -treated rice plants (cv. IR 20) showed a 54% reduction in the length of Sh-R lesions. In three field tests, treatment with P. fluorescens reduced the severity of Sh-R by 20 to 42% in five rice cultivars. Bacterization of rice cultivars with P. fluorescens enhanced plant height, number of tillers, and grain yields from 3 to 160%.

Strains of fluorescent and nonfluorescent bacteria that were isolated from rice rhizospheres of Southern India and showed antagonism towardsRhizoctonia solani were evaluated for biological control of rice sheath-blight (ShB). Efficient strains of bacteria inhibited mycelial growth ofR. solani, affected sclerotial viabilityin vitro and protected IR 20 and TKM 9 rice seedlings from infection byR. solani in greenhouse tests. Pretreatment of sclerotia in bacterial suspensions resulted in reductions in ShB lesion sizes up to 31 to 44% in IR20 and 58 to 74% in TKM 9 rice. In field plots, IR 50 and TKM 9 rice plants raised from bacterized seeds had 65 to 72% less ShB than those plants from untreated seeds.

Systemic chlorotic mottling of trifoliate leaves of soybean cultivars developed following inoculation of unifoliate leaves with strains of Pseudomonas syringae pv. glycinea. This symptom could not be reproduced by application of coronatine, a toxin previously isolated from this bacterium, but resembled systemic symptoms that were produced on bean (Phaseolus vulgaris) by inoculation with P. syringae pv. phaseolicola or by application of toxic principles derived from this pathovar. Attempts to isolate a second toxin from pv. glycinea culture fluids using methods employed for the isolation of phaseolotoxin from pv. phaseolicola were not successful. Evidence was obtained for the presence of inhibitory material that interfered with assays for toxin with Escherichia coli K12 as the test organism. Partial separation of the toxin from the inhibitor and other substances was achieved by high-performance liquid chromatography of culture fluids and further purification was effected by ion exchange, partition, and thin-layer chromatography. Biological activity of the pv. glycinea toxin preparation was similar to a toxin prepared from pv. phaseolicola. Both toxins produced similar systemic symptoms on soybean leaves, inhibited E. coli (reversed by arginine, citrulline, and certain tripeptides), inhibited ornithine carbamoyl transferase, and caused the accumulation of ornithine in soybean leaves, as has been reported for phaseolotoxin. There were small differences between the pv. glycinea and pv. phaseolicola toxins in chromatographic behaviour. It is concluded that, in addition to coronatine, pv. glycinea produces a second toxin that is similar to that produced by pv. phaseolicola.

A total of 117 Pseudomonas fluorescens isolates were obtained from rice rhizospheres collected from 4 states, Tamil Nadu, Andhra Pradesh, Kerala and Karnataka of southern India. The isolates were characterized into biovars and biovar clusters based on the results of tests for 6 primary characters and utilization of 15 carbon sources. Pseudomonas fluorescens biovars identified were, Biovar I with 4 isolates (3%), Biovar II with 16 isolates (15%), Biovar III with 80 isolates (68%), Biovar IV with 2 isolates (2%) and Biovar V with 15 isolates (13%). These results, as well as the cluster analysis, indicate the relative abundance of Biovar III isolates of P. fluorescens in south Indian soils and rice rhizosphere.

Severity of stem-rot disease of peanut caused byRhizoctonia solani was reduced by 54.9 and 68% in plants of two cultivars treated in the greenhouse with antagonistic strains ofPseudomonas fluorescens. These strains were selected based on theirin vitro toxicity to mycelial growth and sclerotial germination ofR. solani. In field experiments, bacterization of peanuts withP. fluorescens resulted in taller plants (by 25.7%) and increased yields (by 59.0%).

Trace amounts of the anthraquinonoid toxic metabolite, 3',4'-dehydro-4'-deoxydothistromin have been identified for the first time, from peanut tissues naturally infected by Cercospora personata. Characteristic uv spectral and chromatographic properties of the metabolite and its tetraacetate as well as the mass spectrum of the latter have established its identity.

Bean seeds challenged withRhizoctonia solani or exposed to natural microflora yielded the isoflavonoid phytoalexins: phaseollin, phaseollidin, phaseollinisoflavan, coumestrol and kievitone. The data suggest that seeds may have the major host-defence factors of a plant.