Pigeonpea Wilt Research Articles

RAPD based molecular diversity analysis of different Fusarium udum Butler isolates of pigeonpea wilt

Genetic diversity in pigeonpea wilt pathogen ( Fusarium udum butler) was analyzed using 10 isolates collected from major pulse growing regions of Gujarat. The genomic DNA extracted from each isolate of Fusarium udum was subjected to polymerase chain reaction using 20 random decamer primers from OPC series. Only 10 of the 20 RAPD primers were selected on the basis of polymorphism for pooled analysis. The 10 earmarked RAPD primers selected from OPC series amplified 67 DNA fragments with size ranging from 157 to 2068bp. Out of these, 47 were polymorphic giving 70 per cent polymorphism. The total number of amplified fragments varied from 3 in OPC-8 to 11 in OPC-4. The average polymorphic bands per primer were 4.7 and per cent polymorphism ranged from 40 in OPC-15 to 100 in OPC-5. The PIC value varied from 0.64 in OPC-8 to 0.88 in OPC-4.

In vitro Efficacy of Fungicides and Bioagents Against Wilt of Pigeonpea Caused by Neocosmospora vasinfecta

Background: Pigeon pea (Cajanus cajan) is one of the important leguminous crop of the tropics and subtropics and is infected by the wilt pathogen Neocosmospora vasinfecta in addition to Fusarium udum. Objective: Hence, the study was undertaken to see the in vitro effect of different fungicides (Thiram 75 WP, Carbendazim 50 WP, Chlorothalonil 75 WP, Metalaxyl MZ 72 WP, Thiram+Cabendazim (2:1), Carbendazim+mancozeb 75 WP, Tricyclazole+Mancozeb 80 WP, Zineb+Hexaconazole 72 WP) and bioagents (Trichoderma harzianum, Pseudomonas fluorescens, Bacillus subtilis) against the pathogen. Methodology: The efficacy of fungicides was assayed by poisoned food technique and of bioagents was assayed by dual culture technique. Results: It was found that among eight fungicides tested carbendazim (0.1%), combination of carbendazim+mancozeb (0.2 %) and thiram+carbendazim 2:1 (0.3%) exhibited cent per cent inhibition of N. vasinfecta, other fungicides were also significant over control. Whereas among bioagents tested, Trichoderma herzianum (50.30%) showed maximum per cent growth inhibition of the pathogen followed by Bacillus subtilis (41.47%). Conclusion: Thus it was proved that the fungicides viz. carbendazim, combinations of carbendazim+mancozeb and thiram+carbendazim as well as bioagent, T. herzianum were effective against Neocosmospora wilt of pigeon pea under in vitro condition.

Mating type genes and genetic markers to decipher intraspecific variability among Fusarium udum isolates from pigeonpea

To ascertain the variability in Fusarium udum (Fu) isolates associated with pigeonpea wilt is a difficult task, if based solely on morphological and cultural characters. In this respect, the robustness of five different genetic marker viz., random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus (ERIC), BOX elements, mating type locus, and microsatellite markers were employed to decipher intra-specific variability in Fu isolates. All techniques yielded intra-specific polymorphism, but different levels of discrimination were obtained. RAPD-PCR was more discriminatory, enabling the detection of thirteen variants among twenty Fu isolates. By microsatellite, ERIC- and BOX-PCR fingerprinting, the isolates were categorized in seven, five, and two clusters, respectively. Cluster analysis of the combined data also showed that the Fu isolates were grouped into ten clusters, sharing 50-100% similarity. The occurrence of both mating types in Fu isolates is reported for the first time in this study. All examined isolates harbored one of the two mating-type idiomorphs, but never both, which suggests a heterothallic mating system of sexual reproduction among them. Information obtained from comparing results of different molecular marker systems should be useful to organize the genetic variability and ideally, will improve disease management practices by identifying sources of inoculum and isolate characteristics.

Two rhizobacterial strains, individually and in interactions with Rhizobium sp., enhance fusarial wilt control, growth, and yield in pigeon pea.

A Pseudomonas aeruginosa strain, RRLJ 04, and a Bacillus cereus strain, BS 03, were tested both individually and in combination with a Rhizobium strain, RH 2, for their ability to enhance plant growth and nodulation in pigeon pea (Cajanus cajan L.) under gnotobiotic, greenhouse and field conditions. Both of the rhizobacterial strains exhibited a positive effect on growth in terms of shoot height, root length, fresh and dry weight, nodulation and yield over the non-treated control. Co-inoculation of seeds with these strains and Rhizobium RH 2 also reduced the number of wilted plants, when grown in soil infested with Fusarium udum. Gnotobiotic studies confirmed that the suppression of wilt disease was due to the presence of the respective PGPR strains. Seed bacterization with drug-marked mutants of RRLJ 04 and BS 03 confirmed their ability to colonize and multiply along the roots. The results suggest that co-inoculation of these strains with Rhizobium strain RH 2 can be further exploited for enhanced growth, nodulation and yield in addition to control of fusarial wilt in pigeon pea.

Wilt epidemiology of pigeonpea (Cajanus cajan (L.) Millsp.) in organic farming system

Pigeonpea (Cajanus cajan (L.) Millsp.) is widely cultivated in hill slopes of Manipur under organic farming system for multipurpose use as fuel, fodder, vegetable, pulse etc. The growing sites where survey was conducted showed different degree of disease incidence and severity of wilt in pigeonpea caused by Fusarium udum. Maximum disease incidence (58.77 % ;early maturing (V1 ) & 42.74% ;late maturing (V2)) and severity (55.22% ; V1 & 40.96% ; V2) were found at sampling sites (Motbung ; V1 & Kanglatongbi ; V2) where pigeonpea is grown continuously. Disease progress is slow during the early phases of growth but accelerates during the flowering and podding stage. Rate of infection was highest in flowering and podding stage and there was slight variation in area under disease progress curve between the sampling sites. Significant positive and negative correlations were observed between disease incidence and severity and meteorological factors.

Fusarial wilt control and growth promotion of pigeon pea through bioactive metabolites produced by two plant growth promoting rhizobacteria

The bioactive metabolites produced by two plant growth promoting rhizobacteria strains, a Pseudomonas aeruginosa strain RRLJ 04 and a Bacillus cereus strain BS 03, which showed growth promotion and disease control in pigeon pea against Fusarium udum, were isolated and screened for their efficacy to control fusarial wilt of pigeon pea under gnotobiotic and nursery condition. Bioactive metabolites viz., BM 1 and BM 2 from RRLJ 04 and BM 3 from BS 03 also showed in vitro antibiosis against F. udum. Seeds treated with 50 μl seed⁻¹ of BM 1, 30 μl seed⁻¹ of BM 2 and 70 μl seed⁻¹ of BM 3 and grown in pathogen infested soil showed suppression of wilt disease besides growth enhancement. Per cent disease control was 90 % with BM 2 application as compared to 87 and 83 %, respectively in BM 1 and BM 3 after 90 days of growth. BM 2 treated plants were more resistant to the pathogen as compared to the other fractions tested. Mycelial dry weight was found to be reduced on treatment with the bioactive metabolites. Formation of chlamydospore-like structures was observed in the pathogen mycelium treated with BM 3. The analytical studies confirmed that two of these metabolites are phenazine derivatives.

Genetic diversity of Fusarium wilt races of pigeonpea in major regions of India.

Fusarium wilt is a serious fungal disease in pigeonpea (Cajanus cajan) which causes severe yield loses (up to 90%). Genetic diversity in pigeonpea wilt pathogen [Fusarium udum (fud)] was characterised using 14 isolates collected from major pulse growing regions of India. Twenty four RAPD primers generated a total of 226 bands (ranging 0.3 to 3.0 kb) in F. udum with an average of 9.4 bands per primer and a total of 27 alleles were produced by twelve SSR primers with an average of 2.25 alleles per marker. All isolates amplified a single band ranging from 100 to 450 bp. The universal ITS primer pair amplified 650 bp bands in all fourteen fud isolates, while significant length polymorphism was obtained only when analysed by restriction digestion with EcoRI and Hind III enzymes. Cluster analysis of ITS-RFLP grouped all 14 Fud isolates into three major clusters. Cluster analysis using various markers showed the grouping of Fusarium isolates strictly according to their cultural characteristics and degree of pathogenicity and not the geographical origin. This information will be helpful for pathologists and plant breeders to design effective resistance breeding programmes in pigeonpea taking into account the diversity in wilt pathogen.

Management of Pigeonpea Wilt Caused by Fusarium udum Butler through Integrated Approaches

Integrated Disease Management (IDM) approach was carried out to combat pigeonpea wilt with a combination of fungicides, bio agents, organic amendments and different cropping systems in kharif seasons of four years viz., 2006, 2007, 2008 and 2009. Based on the performance, four treatments were identified as best practices for the management of pigeonpea wilt viz., carbendazim seed treatment @ 2 g/kg of seeds + Trichoderma viride @ 2.5 kg/ha in FYM @ 50 kg / ha recorded lowest mean wilt incidence of 11.38 per cent with highest mean yield of 969.18 kg/ha, followed by carbendazim seed treatment @ 2g/Kg of seeds + ZnSo4 @ 25 kg/ha which recorded mean wilt incidence of 11.75 per cent with mean yield 951.25 kg/ha. However, Trichoderma viride seed treatment @ 5g/kg of seeds + Trichoderma viride @ 2.5 kg/ha in FYM @ 50 kg/ha which recorded mean wilt incidence of 11.97 per cent and yield of 929.63 kg/ha and pigeonpea intercrop with sorghum @ 1:1 recorded mean wilt incidence of 17.62 per cent and yield of 632.18 kg/ha were also found effective in controlling wilt incidence and increasing yield compared to untreated control which recorded highest wilt incidence of 53.04 per cent and lowest yield (314.95 kg/ha).

Identification of dominant and recessive genes for resistance to Fusarium wilt in pigeonpea and their implication in breeding hybrids

Fusarium wilt is an important disease of pigeonpea [Cajanus cajan (L.) Millsp.] and it can cause severe yield losses. Chemical control of this disease is difficult and expensive; therefore, cultivation of resistant varieties/hybrids is the most efficient strategy for enhancing the production. In the present study, by using a wilt susceptible cytoplasmic-nuclear male-sterile line and four wilt resistant fertility restorers, one dominant and one recessive gene with dominant suppressive epistatic effects were found responsible for controlling resistance to Fusarium wilt. Considering the annual losses and wide spread nature of wilt diseases in pigeonpea, it is imperative that all the inbred and hybrid cultivars have high level of resistance to this disease. The presence of dominant gene for resistance will increase the efficiency of breeding wilt resistant cultivars because it will yield greater proportion of resistant genotypes in segregating generations. In hybrid breeding also, the presence of dominant gene for wilt resistance will be an advantage. The transfer of this gene in female hybrid parents will ease the breeding of wilt resistant hybrids because this will allow the use of both wilt resistant as well as susceptible restorers in generating wilt resistant hybrid combinations.

Inheritance of resistance to Fusarium wilt and yield traits in pigeonpea

Fusarium wilt is the main pigeonpea production constraint in Malawi. The purpose of the study was to understand the nature and mechanism of inheritance of F. wilt resistance, yield and secondary traits in pigeonpea. 48 crosses were generated in a 12 lines × 4 testers mating scheme. Some F1 plants were selfed for segregation analysis for inheritance pattern of resistance, while others were evaluated for resistance, yield and secondary traits. There were significant variations among F1 plants for F. wilt, days to 50 % flowering, seed/pod, and number of secondary branches. Specific combining ability (SCA) effects were predominant for F. wilt, days to 50 % flowering and number of secondary branches. The general combining ability (GCA) effects, mainly due to maternal genotypes, were preponderant for yield and other secondary traits. The significance of GCA and SCA effects suggested that variations were due to additive gene action in both the testers and parental lines arising from their interactions, and the dominance effects due to interactions of the parental lines. The χ2 analysis suggested dominant patterns of inheritance for wilt in most of the F2 populations. The segregation ratios of 3:1, 15:1, and 9:7 suggested the involvement of single or two independent/complementary dominant genes in the test donors. Involvement of a few genes governing wilt resistance suggested the ease of breeding for this trait. Pedigree breeding method would be recommended for incorporating various traits in pigeonpea.

Combined use of Streptomyces sp. A6 and chemical fungicides against fusarium wilt of Cajanus cajan may reduce the dosage of fungicides required in the field

Biological control agents offer one of the best alternatives to reduce the use of pesticides. This investigation studied the tolerance to fungicides and integrated use of the potential biocontrol agent Streptomyces sp. A6 for control of Fusarium wilt of pigeon pea, Cajanus cajan. Streptomyces sp. A6 exhibited strong tolerance towards most of the fungicides used in the study at concentrations higher than those recommended for field applications. The isolate showed enhanced growth and mycolytic enzyme production in the presence of sulphur, mancozeb, carbendazim, fosetyl aluminium and triadimefon. The fungicides mancozeb, sulphur and carbendazim were selected for further studies. Effective concentrations (EC 50 values) of the test fungicides that reduced Fusarium spore germination and fungal biomass by 50% were determined. Similarly, the EC 50 for inhibiting fungal spore germination and reducing fungal biomass to 50% by Streptomyces sp. A6 and culture filtrate (CF) were also determined. Combining the EC 50 dose of the culture and CF with test fungicides was found to be more effective for controlling Fusarium infection in C. cajan compared to the sum of the effects of the individual treatments. Such combined use of biocontrol agent with fungicides can reduce the dosage of toxic fungicides in agricultural fields, thereby reducing environmental risks. Tolerance and synergistic interaction of Streptomyces sp. A6 with frequently used fungicides suggested its potential in integrated pest management. To the best our knowledge, this is the first extensive study on integrated use of Streptomyces species with fungicides.

Rapid detection and quantification of Fusarium udum in soil and plant samples using real-time PCR

Real-time PCR based detection assay was developed for Fusarium udum, causing vascular wilt of pigeonpea. The Histone-3 gene of F. udum was targeted to design species-specific primers and probe. A comparative study was undertaken to develop a reliable and reproducible procedure for detection and quantification of F. udum from diverse samples. The sensitivity and specificity of oligonucleotides were evaluated through dot blot hybridization, standard and real-time PCR assays. The probe HFUSP showed high degree of sensitivity for DNA obtained from pure cultures of F. udum to that of environmental DNA samples. The qPCR assay specifically differentiated the F. udum from closely related species of Fusarium, other test microbes and environmental samples. The single melting curve at 84.17 and a monomorphic band of 200 bp indicates the specificity and authenticity of the PCR assays. Thus, real-time PCR assay can be used as a rapid and effective procedure that can detect minute amounts of F. udum from complex environments. Therefore, the real-time PCR assay demonstrated in the present study can be successfully used for detection and monitoring of F. udum for early infection and disease epidemiology of vascular wilt of pigeonpea.

Simple sequence repeat‐based diversity in elite pigeonpea genotypes for developing mapping populations to map resistance to Fusarium wilt and sterility mosaic disease

With 1 figure and 3 tables AbstractIn order to maximize polymorphism in the mapping populations for mapping loci for Fusarium wilt (FW) and sterility mosaic disease (SMD) resistance in pigeonpea, a set of 32 pigeonpea lines were screened for polymorphism with 30 microsatellite or simple sequence repeat markers. A total of 23 marker loci showed polymorphism with 2–4 alleles and the polymorphism information content for these markers ranged from 0.12 to 0.65 with an average of 0.43 per marker. High number of polymorphic markers, higher genetic dissimilarity coefficient and contrasting phenotypic data taken into consideration and five parental combinations were identified and crosses initiated for developing five genetically diverse mapping populations. Of these crosses, one cross segregates for FW resistance, two for SMD resistance and the remaining two crosses segregate for resistance to both FW and SMD. Development of mapping populations is in progress for mapping loci for resistance to FW and SMD in pigeonpea.

Mass multiplication and formulations of biocontrol agents for use against fusarium wilt of pigeonpea through seed treatment

Trichoderma harzianum and Pseudomonas fluorescens are the two major fungal biocontrol agents found in the soil and the rhizosphere of various crop systems. We used different agricultural waste bases such as wheat bran, rice bran, paddy straw and neem cake for mass-multiplying T. harzianum and P. fluorescens. Ten isolates each of T. harzianum and P. fluorescens were isolated from rhizosphere soil samples collected from various pigeonpea-growing fields. We evaluated the efficacy of these isolates, both in increasing seed quality variables of pigeonpea and in inhibiting the mycelial growth of Fusarium udum Butler. Trichoderma harzianum isolate 4 and Pseudomonas fluorescens isolate 3 were selected and mass-multiplied using different agricultural waste products as bases. Among the latter, boiled rice bran was found to increase the growth of both biocontrol agents. Talc and sodium alginate formulations of mass-multiplied biocontrol agents were prepared and evaluated for their affects against fusarium wilt under greenhouse conditions. Fresh cultures of both biocontrol agents were found to increase seedling emergence and reduce fusarium wilt disease incidence when compared to the control and the formulations.

Evaluation of Trichoderma species against Fusarium udum Butler Causing Wilt of Pigeonpea

One isolate each of Trichoderma viride, Trichoderma harzianum and Trichoderma virens were evaluated against twelve isolates of Fusarium udum collected from different districts of Bihar, Jharkhand and West Bengal by dual culture technique. All the bioagents inhibited the growth of F. udum in vitro . Dholi isolate of T. harzianum inhibited the growth of F. udum isolates Fu-5, Fu-12, Fu-14, Fu-21, Fu-24, Fu-27 and Fu-43 by 40.8, 74.5 , 49.4, 59.1, 60.5, 40.8 and 57.8 per cent, respectively and proved superior to other antagonists. T. viride was found to have potential in inhibiting the radial growth of F. udum isolates Fu-29, Fu-37, Fu-49 and Fu-57 while T. virens was more effective in inhibiting the growth of Fu-61. Combined application of T. harzianum and T. viride as seed treatment and soil application gave minimum disease incidence (26.4%) and maximum disease control (56%) followed by 54% disease control in T. harzainu m seed treatment and soil application. However, these two treatments were on par with each others. The results also revealed that soil application of bioagents was more effective than seed treatment.

Induction of systemic resistance against fusarial wilt in pigeon pea through interaction of plant growth promoting rhizobacteria and rhizobia

A Bacillus cereus strain BS 03 and a Pseudomonas aeruginosa strain RRLJ 04 were studied for their effect on induction of systemic resistance against Fusarium udum wilt in pigeon pea, both individually and in combination with a rhizobial strain RH 2. Split root experiments confirmed that plants with combination treatment of PGPR and Rhizobium can survive longer than individual treatments and control. An increased level of defense-related enzymes, viz., l-phenylalanine ammonia lyase (PAL), peroxidase (POX) and polyphenol oxidase (PPO), were also recorded in co-inoculated plants. Production of β-1,3-glucanase and polymethyl galacturonase by the pathogen in culture medium was also sharply reduced in presence of both the PGPR strains. An increased level of phenol content with a decreased level of nitrogen content was recorded in the leaves of bacterized plants grown in pathogen infested soil. Presence of BS 03 in the culture medium stimulated the production of chlamydospore-like structures in the fungal mycelium. Such structures were not observed in case of RRLJ 04 treatment but the dry weight of mycelium was reduced significantly. Fusaric acid production by the pathogen was also drastically reduced in the presence of the PGPR strains. The results promise the combined use of PGPR and rhizobia for induction of systemic resistance against fusarial wilt in pigeon pea.

Impact of a New Source of Resistance to Fusarium Wilt in Pigeonpea

AbstractPigeonpea is an important grain legume grown by smallholder farmers in Southern Africa. Fusarium wilt, caused by the fungal pathogen Fusarium udum Butler, is the major disease limiting pigeonpea production in the region. This study was designed to evaluate the reaction to fusarium wilt as well as agronomic performance of new elite pigeonpea germplasm in three different countries during the 2001/2002 cropping season using wilt‐sick plots. Per cent incidence of fusarium wilt (%FW), grain size and yield, were measured. The genotype ICEAP 00040 consistently showed a high (<20.0%) level of resistance to the disease in all three countries. In contrast, %FW score for the susceptible genotype ICEAP 00068 was 87.5, 92.0 and 90.9% in Kenya, Malawi and Tanzania, respectively. The grain size obtained for ICEAP 00040 at Ngabu (Malawi) was 25.0% larger than that at each of the remaining locations indicating environmental influence on this trait. At all the three locations, ≥1.5 ton/Ha of grain yield was obtained for ICEAP 00040 compared with <1.0 ton/Ha for ICEAP 00068. In 2003, this improved resistant genotype (ICEAP 00040) was released for commercial production and will be useful as a good source of resistance in pigeonpea genetic improvement programs in the region.

Effect of genotype and root colonization in biological control of fusarium wilts in pigeonpea and chickpea by Pseudomonas aeruginosa PNA1.

Pseudomonas aeruginosa PNA1, an isolate from chickpea rhizosphere in India, protected pigeonpea and chickpea plants from fusarium wilt disease, which is caused by Fusarium oxysporum f.sp. ciceris and Fusarium udum. Inoculation with strain PNA1 significantly reduced the incidence of fusarium wilt in pigeonpea and chickpea on both susceptible and moderately tolerant genotypes. However, strain PNA1 protected the plants from fusarium wilt until maturity only in moderately tolerant genotypes of pigeonpea and chickpea. Root colonization of pigeonpea and chickpea, which was measured using a lacZ-marked strain of PNA1, showed tenfold lower root colonization of susceptible genotypes than that of moderately tolerant genotypes, indicating that this plant-bacteria interaction could be important for disease suppression in this plant. Strain PNA1 produced two phenazine antibiotics, phenazine-1-carboxylic acid and oxychlororaphin, in vitro. Its Tn5 mutants (FM29 and FM13), which were deficient in phenazine production, caused a reduction or loss of wilt disease suppression in vivo. Hence, phenazine production by PNA1 also contributed to the biocontrol of fusarium wilt diseases in pigeonpea and chickpea.

Effect of soil and seed application of Trichoderma harzianum on pigeonpea wilt caused by Fusarium udum under field conditions

The efficacy of the biological control agent Trichoderma harzianum was tested on various levels of Fusarium udum, the pigeonpea wilt pathogen. Field plots infested with the pathogen at three inocula levels (log 3.04, log 4.98 and log 5.34 colony-forming units (cfu)/g of soil) were identified. Trichoderma harzianum was applied as a seed treatment (10 and 20 g/kg seed) and as a soil amendment (10 and 20 g/9 m 2). The Trichoderma population increased to more than 10 8 cfu/g soil by 60 days in treated plots, whereas for seed treatments, fungal population reached a maximum of 10 4.62 cfu/g soil within 45 days, and thereafter started to decline. At log 3.04 pathogen concentration, soil amendment, with T. harzianum at 10 and 20 g gave 42.9% and 61.5% disease control, respectively. Seed treatments resulted in less than 30% disease control when compared to the check. The disease control achieved with fungicide seed treatment was insignificant. At log 4.98 and log 5.34 pathogen concentrations, disease control ranged between 22% and 35.3% for soil treatments. Even at the highest pathogen density (log 5.34), soil amendment with T. harzianum at 10 g gave about 30% disease reduction. In seed treatment plots, disease control ranged between 4.36% and 13.7%. In general, soil application of T. harzianum was found to be more effective than seed treatment for disease suppression. These results suggest the need to augment soil applications of T. harzianum for obtaining effective control of pigeonpea wilt.

Chitin-supplemented formulations improve biocontrol and plant growth promoting efficiency of Bacillus subtilis AF 1.

Formulations of a chitinolytic biocontrol and a plant growth promoting Bacillus subtilis AF 1 were prepared in peat, in peat supplemented with either 0.5% chitin or Aspergillus niger mycelium, or in spent compost obtained from Agaricus bisporus cultivation and were evaluated for biocontrol of two fungal pathogens and plant growth promoting activities on pigeon pea and groundnut. A steady increase in cell numbers of introduced B. subtilis AF 1 was observed in all the formulations at 30 degrees C. The increase in cell numbers was about 5.0 log units. Peat or spent compost inoculated with physiologically active and dormant states of B. subtilis AF 1 showed different time period requirements to attain maximum cell numbers. The presence of chitin or A. niger (in peat) or A. bisporus (in spent compost) as supplement in the carrier material improved the multiplication of B. subtilis AF 1. When used as seed treatments, formulations of AF 1 in peat supplemented with chitin or chitin-containing materials showed better control of A. niger (causing crown rot of groundnut) and Fusarium udum (causing wilt of pigeon pea) than AF 1 culture alone, in both groundnut and pigeon pea. Bacillus subtilis AF 1 formulations promoted seed germination and biomass of both groundnut and pigeon pea even under pathogen pressure. Survival of AF 1 on fresh culture-treated and formulation product-treated plants was similar in pathogen-infested soil.