Fusarium wilt caused by
f. sp.
(
) is the most devastating disease of lentil present worldwide. Identification of multi-race fusarium wilt resistance genes and their incorporation into existing ...cultivars will help to reduce yield losses. In the present study, 100 lentil germplasms belonging to seven lentil species were screened against seven prevalent races of
, and accessions IC201561 (
subsp.
, EC714243 (
. subsp.
, and EC718238 (
were identified as resistant. The typical R gene codes for the nucleotide-binding site and leucine-rich repeats (NBS-LRR) at the C terminal are linked to either the Toll/interleukin 1-like receptor (TIR) or coiled coil (CC) at the N terminal. In the present study, degenerate primers, designed from the NBS region amplifying the P-loop to the GLPLA motif, isolated forty-five resistance gene analogues (RGAs) from identified resistant accessions. The sequence alignment identified both classes of RGAs, TIR and non-TIR, based on the presence of aspartate (D) and tryptophan (W) at the end of the kinase motif, respectively. The phylogenetic analysis grouped the RGAs into six classes, from LRGA1 to LRGA6, which determined the diversity of the RGAs present in the host. Grouping of the RGAs identified from
, LnRGA 2, 9, 13 with I2 revealed the structural similarity with the fusarium resistance gene. The similarity index ranged from 27.85% to 86.98% among the RGAs and from 26.83% to 49.41% among the known R genes, I2, Gpa2, M, and L6. The active binding sites present along the conserved motifs grouped the RGAs into 13 groups. ADP/ATP, being the potential ligand, determines the ATP binding and ATP hydrolysis activity of the RGAs. The isolated RGAs can be used to develop markers linked to the functional R gene. Furthermore, expression analysis and full-length gene isolation pave the path to identifying the molecular mechanism involved in resistance.
Plant diseases cause serious economic losses of agriculture production worldwide. Rapid, accurate and reliable diagnostic methods are required to alleviate the detection of fungal plant pathogens to ...prevent their spread and achieve effective management. This study was aimed to develop fast, reliable and highly sensitive diagnostics to detect fungal plant pathogens for quarantine processing, safe exchange and conservation of germplasms of pulse crops. Multiplex and real time PCR assays were developed for detection of
Rhizoctonia solani
,
Macrophomina phaseolina
,
Ascochyta rabiei
,
Alternaria alternata
,
A. tenuissima
,
Fusarium oxysporum
f. sp.
ciceris
,
Sclerotium
(
Athelia
)
rolfsii
,
Sclerotinia sclerotiorum
,
Pseudocercospora cruenta
and
Cercospora canescens
causing various diseases in pulse crops. Twenty-two sets of primers from various genomic regions such as cytochrome oxidase subunit (COX 1), internal transcribed spacer region (ITS), translation elongation factor-1 alpha (TEF-1α), large subunit (LSU), small subunit (SSU) and β-tubulin as well as two SCAR primers from RAPD profile were designed. The developed markers proved to be species-specific and validated against other fungal plant pathogens associated with pulses for cross-reactivity. The markers proved highly sensitive during conventional and qPCR analysis. Duplex PCR assays for
R. solani
and
M. phaseolina
;
C. canescens
and
P. cruenta
;
A. alternata
and
A. tenuissima
; and a quadruplex PCR assay for
A. rabiei
,
S. sclerotiorum
,
S. rolfsii
and
F. oxysporum
f. sp.
ciceris
were developed and validated for simultaneous detection of these pathogens in a single reaction. The assays developed in the present study were able to detect and identify major fungal plant pathogens causing disease in pulse crops.
Fusarium wilt (
Fusarium oxysporum f. sp.
ciceris (Padwick) Matuo and K. Sato) is one of the major yield limiting factors of chickpea (
Cicer arietinum L.). For eco-friendly and sustainable ...management of the disease, 10 isolates belonging to three species of
Trichoderma (
Trichoderma viride,
Trichoderma harzianum, and
Trichoderma virens) were evaluated against four isolates of the pathogen representing four different races commonly prevalent in India. Dharwad (race 1), Kanpur (race 2), Ludhiana (race 3), and Delhi (race 4) isolates of
F. oxysporum f. sp.
ciceris were included in the study. The isolates of
Trichoderma species were evaluated against the pathogen in dual culture and through production of volatile and non-volatile inhibitors.
T. viride isolated from Ranchi followed by
T. harzianum (Ranchi) and
T. viride isolated from Delhi inhibited maximum mycelial growth of the pathogen. They also enhanced seed germination, root and shoot length, and decreased wilt incidence under green house condition. The isolates proved potential
in vitro tests were evaluated along with other bioagents individually and in combination with carboxin under wilt sick field during 2002/03, 2003/04, and 2004/05 cropping season in randomized block design in three replications. Species of
Trichoderma were found superior to
Bacillus subtilis and Kalisena™ a commercial formulation of
Aspergillus niger. The efficacy of
Trichoderma species was enhanced in combination with carboxin. The integration of
T. harzianum (10
6 spores/ml/10
g seed) and carboxin (2
g
kg
−1 seed) for seed treatment was the best which enhanced seed germination by 12.0–14.0% and grain yields by 42.6–72.9% and reduced wilt incidence (44.1–60.3%) during experimentations.
Soybean is one of the most important crops grown worldwide and accounting for significant global trade including transgenic soybean. The crop is attacked by several seed-borne fungal pathogens and ...some of them are of quarantine concern for India. Keeping in view of the risks associated with movement of soybean seeds, sensitive and reliable molecular diagnostics have been developed for precise and simultaneous detection of three pathogens of quarantine concern for India namely,
Diaporthe phaseolorum
(stem blight),
D. longicolla
(seed decay),
Peronospora manshurica
(downy mildew), along with
Macrophomina phaseolina
causing dry root rot. The targeted pathogens after isolation from imported transgenic and non-transgenic soybean seeds were identified. Quadruplex and qPCR assays were developed targeting the sequences of different genes such as
Histone-3
for detection of
D. longicolla
and
M. phaseolina.
The markers DlHisF2&R2 and MpHisF1&R1 produced 265 and 309 bp amplicons for
D. longicolla
and
M. phaseolina
, respectively.
Actin
gene based marker DpActF1&R2 was developed for
D. phaseolorum
which provided 113 bp amplicon whereas,
COX2
based marker PmCoxF2&R2 was developed for
P. manshurica
with amplified product of 152 bp. During qPCR analysis, these markers proved highly specific and sensitive for detection of these pathogens up to 0.1 pg of template DNA. Quadruplex PCR protocol was also developed by combining these specific markers which could distinguish all the targeted pathogens simultaneously in a single reaction. The developed diagnostic protocols are extremely valuable for quarantine clearance and to ensure the safe transboundary exchange and healthy conservation of germplasm in the National Genebank.
Field experiments were conducted during the rainy seasons of 2009 and 2010 for the management of the major diseases of mungbean, namely, wet root rot (Rhizoctonia solani), cercospora leaf spots ...(Cercospora canescens and Pseudocercospora cruenta) and yellow mosaic (Mungbean Yellow Mosaic Virus) by using different combinations of an insecticide, fungicide, and bio-formulation as seed treatment, with or without foliar sprays. A combination of seed treatment with thiamethoxam (Cruiser™) at 4 g kg−1, carboxin (Vitavax™) at 2 g kg−1 and Pusa 5SD (Trichoderma virens) at 4 g kg−1 followed by simultaneous foliar sprays of thiamethoxam (Actara™) 0.02% and carbendazim (Bavistin™) 0.05% at 21 and 35 days after sowing resulted in the highest seed germination and grain yield in mungbean with the lowest intensities of cercospora leaf spots and mungbean yellow mosaic, and moderate incidence of wet root rot. The lowest whitefly population was also observed in this treatment during all stages of the crop. The treatment combinations having Pusa 5SD as seed treatment provided the lowest wet root rot incidence. Two sprays were superior to single spray for all variables recorded, but in combination with seed treatment, single spray was found to be more cost effective as it obtained the highest return per rupee of input. Use of T. virens based bio-formulation Pusa 5SD with insecticide thiamethoxam has been effectively demonstrated for the first time along with fungicides Bavistin and Vitavax for the management of the major diseases of mungbean.
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► An integrated management strategy was developed for major diseases of mungbean. ► A combined seed treatment with thiamethoxam, carboxin and Pusa 5SD proved effective. ► Combined foliar sprays of thiamethoxam and carbendazim at 21 and 35 DAS proved effective. ► The combination of seed treatment and two foliar sprays proved to be the best. ► A combination with seed treatment with single spray was found to be more cost effective.
Genetic diversity of 70 isolates of Fusarium oxysporum f. sp. ciceris originated from various states of India representing eight races causing wilt in chickpea (Cicer arietinum) was analyzed using ...translation elongation factor-1α (TEF-1α), β-tubulin, and internal transcribed spacer (ITS) gene regions. TEF-1α, β-tubulin, and ITS gene-specific markers produced ~720-, ~500-, and ~550-bp amplicons, respectively, in all the isolates of the pathogen. A phylogenetic tree constructed from the sequences generated in the present study along with the sequences of foreign isolates of Fusarium species available in NCBI database sharing more than 90 % nucleotide sequence similarity grouped the isolates into two major clusters. Most of the isolates of the present study showed more or less similar grouping pattern in case of the three gene sequences. Each group had the isolates representing different races as well as place of origin indicating low level of diversity among the isolates in respect of these gene sequences. Except TEF-1α, the groups generated by β-tubulin and ITS gene sequences did not correspond to the state of origin and races of the pathogen. However, the groups of TEF-1α partially corresponded to the place of origin as well as races of the pathogen. The isolates did not show any race-specific grouping patterns; however, most of the isolates representing race 1 clustered separately.
Ascochyta blight of chickpea is caused by
Ascochyta rabiei
(Pass.) Labr. which is primarily seedborne. For rapid detection and precise identification of
A. rabiei
, a sequence-characterized amplified ...region (SCAR) marker was developed for detection of genomic DNA and infected plant DNA. An SSR primer amplified monomorphic band was cloned in pGEM®-T easy vector and sequenced. The best primer pair was selected and validated on
A. rabiei
. The specificity and sensitivity of the SCAR-based marker designated as MBAR was evaluated using conventional PCR and real-time PCR. The marker produced consistently an amplicon size of 196 bp in all
A. rabiei
isolates tested. The sensitivity of the marker was 0.1 ng of genomic fungal DNA and 0.5 ng of plant DNA by conventional PCR and 0.5 pg of
A. rabiei
DNA and 1.0 pg of plant DNA by real-time PCR. This is the first SCAR marker having high specificity and sensitivity towards
A. rabiei
. The marker may be useful in detecting the pathogen before the disease appearance and in plant quarantine program to detect the pathogen in seed lots.
Web blight/wet root rot caused by
Rhizoctonia solani
is one of the major constraints for mung bean (
Vigna radiata
) production. Growing of resistant varieties and use of biocontrol agents are the ...feasible options available to manage the disease. The present study was conducted to determine the variation in the expression of various defense-related genes in susceptible and resistant mung bean varieties in response to biocontrol agent
Trichoderma virens
and
R. solani
interactions. The primers were designed using sequences of defense-related genes, namely PR 10, epoxide hydrolase (EH), catalase and calmodulin available in NCBI database and evaluated against cDNA obtained from both susceptible and resistant mung bean plants at 1–4 days post-inoculation (dpi) with the test pathogen
R. solani
and biocontrol agent
T. virens
using conventional PCR and qPCR analyses.
R. solani
inoculation upregulated the mean expression of PR 10 and calmodulin in susceptible and resistant varieties, respectively, whereas downregulated in the rest of the treatments. Quantitative PCR analysis showed that except catalase in the susceptible variety, which is downregulated, the expression of PR 10, EH, catalase and calmodulin was upregulated in both resistant and susceptible varieties in response to
T. virens
alone and in the presence of
R. solani
. In general, the expression of PR 10 and calmodulin was highest at 1 dpi whereas EH and catalase expression were maximum at 4 dpi. The application of
T. virens
suppressed the development of disease in the presence of
R. solani
in both susceptible and resistant varieties with more pronounced effect in resistant variety. Thus, the application of biocontrol agent
T. virens
upregulated the expression of defense-related genes and reduced disease development.
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