Fusarium Wilt Of Chickpea Research Articles

Pathogenicity of indole-3-acetic acid producing fungus Fusarium delphinoides strain GPK towards chickpea and pigeon pea

An indole-3-acetic acid (IAA) producing fungal strain was isolated from chickpea grown rhizospheric soil samples. Based on morphological and Internal Transcribed Spacer (ITS) region sequence analysis the new isolate was identified as Fusarium delphinoides. The Fusarium delphinoides strain produces and secretes IAA in-vitro as identified by HPLC and Mass spectrometry. The IAA production is dependent on tryptophan (Trp) as a nitrogen source in the medium. The IAA production is influenced by growth conditions such as pH of the medium, concentration of Trp and the nature of the carbon source. Additional nitrogen sources repress Trp dependent IAA production. Glucose and Trp served as the best carbon and nitrogen sources respectively. Pathogenicity of Fusarium delphinoides towards the plants was tested by electrolyte, nutrient leakage analysis and also by scoring the disease symptoms. Two cultivars of chickpea (ICCV-10 and L-550) and two cultivars of pigeon pea (Maruti and PT-221) were assessed for the pathogenicity by inoculating with spores of Fusarium delphinoides. The inoculation induced symptoms of Fusarium wilt as in the case of Fusarium oxysporum f. sp. ciceris (FOC), a known pathogen causing Fusarium wilt in chickpea. Electrolyte and nutrient leakage from the infected plants were used to assess the resistance, tolerance (moderately resistance) and susceptibility of the plants to the infection. Based on the results, both the pigeon pea cultivars (Maruti and PT-221) were rated as resistant, and ICCV-10 was rated as a tolerant cultivar of chickpea. However, chickpea cultivar L −550 was found to be a susceptible host for infection by Fusarium delphinoides. These results suggest that Fusarium delphinoides, which belongs to the Fusarium dimerum species group, is an IAA producing plant pathogen and causes wilt in chickpea. Further, along with pathogenicity tests, electrolyte and nutrient leakage analysis can be used to assess the pathogenicity of pathogenic fungi.

Effect of mustard green manure and dried plant residue on chickpea wilt (Fusarium oxysporum f.sp. ciceris)

Pot experiments were carried out in the green house at Amhara Regional Agriculture Research Institute (ARARI) Bahirdar, Ethiopia, to evaluate the potential of Brassica carinata cultivars, namely Holleta-l, S-67 and Yellow Dodola in 2007 and 2008. The effect of B. carinata (Ethiopian mustard) cultivars Holleta-1, S-67 and Yellow Dodola as green manure and Holleta-1 as dried plant residue on chickpea fusarium wilt (Fusarium oxysporum f.sp. ciceris) was studied. Six rates of green manure and dried plant residue (0, 20, 40, 60, 80 and 100 g) each per kg of pathogen-infested soil were used in the experiments. Infested soil without B. carinata cultivars amendment as a control and susceptible check variety JG-62 without amendment was used in the experiments. In the experiments, the treatments were arranged in randomised complete block design in three replications and repeated twice. Data on seedling emergence, wilt incidence, fresh weight and dry weight were collected. The amendments of infested soil with B. carinata cultivars green manure and dried plant residue reduced the incidence of chickpea fusarium wilt. The incorporation of the green manure Holleta-1, S-67 and Yellow Dodola at 20–100 g/kg of infested soil was effective in reducing wilt incidences on chickpea. However, the incorporation of Yellow Dodola at 80 and 100 g green manure per kg of infested soil were the best combination in reducing significantly wilt incidence. The application of the dried plant residue at 20–100 g/kg of infested soil was effective in reducing wilt incidences on chickpea. However when applied dried plant residue at 60, 80 and 100 g green manure per kg of infested soil were better in reducing wilt incidence as compared to 20 and 40 g/kg of infested soil. The three cultivars green manure incorporated at different level of doses affected the influence of fusarium wilt on the fresh and dry weight respectively. The use of Holleta-1 green manure at 20–100 g/kg of infested soil significantly reduced disease incidence in the range of 20.0–33.3%. Green manure amendment S-67 significantly reduced disease incidence in the range of 20.0–46.6%. Yellow Dodola reduce disease incidence with 26.7–60%. The dried plant residue incorporated at different level influence fusarium wilt. The application of Holleta-1 dried plant residue at 20–100 g/kg of infested soil reduced disease incidence in the range 20.0–26.7%. The results imply the potential of using B. carinata green manure and dried plant residue as cultural management components in chickpea fusarium wilt disease management.

Evaluation of actinomycete isolates obtained from herbal vermicompost for the biological control of Fusarium wilt of chickpea

A total of 137 actinomycetes cultures, isolated from 25 different herbal vermicomposts, were characterized for their antagonistic potential against Fusarium oxysporum f. sp. ciceri (FOC) by dual-culture assay. Of the isolates, five most promising FOC antagonistic isolates (CAI-24, CAI-121, CAI-127, KAI-32 and KAI-90) were characterized for the production of siderophore, cellulase, protease, hydrocyanic acid (HCN), indole acetic acid (IAA) and antagonistic potential against Rhizoctonia bataticola, which causes dry root rot in chickpea (three strains viz. RB-6, RB-24 and RB-115) and sorghum (one strain). All of the five FOC antagonistic isolates produced siderophore and HCN, four of them (except KAI-90) produced IAA, KAI-32 and KAI-90 produced cellulase and CAI-24 and CAI-127 produced protease. In the dual-culture assay, three of the isolates, CAI-24, KAI-32 and KAI-90, also inhibited all three strains of R. bataticola in chickpea, while two of them (KAI-32 and KAI-90) inhibited the tested strain in sorghum. When the FOC antagonistic isolates were evaluated further for their antagonistic potential in the greenhouse and wilt-sick field conditions on chickpea, 45–76% and 4–19% reduction of disease incidence were observed, respectively compared to the control. The sequences of 16S rDNA gene of the isolates CAI-24, CAI-121, CAI-127, KAI-32 and KAI-90 were matched with Streptomyces tsusimaensis, Streptomyces caviscabies, Streptomyces setonii, Streptomyces africanus and an identified species of Streptomyces, respectively using the BLAST searching. This study indicated that the selected actinomycete isolates have the potential for biological control of Fusarium wilt disease in chickpea.

Control of chickpea wilt (Fusarium oxysporum f.sp. ciceris) using Trichoderma spp. in Ethiopia

Thirty-two Trichoderma isolates were collected from soils grown with chickpea in central highlands of Ethiopia. The eight isolates were identified by CAB-International as Trichoderma harzianum, T. koningii and T. pseudokoningii. In in vitro tests, all Trichoderma isolates showed significant (P < 0.05) differences in their colony growth and in inhibiting the colony growth of Fusarium oxysporum f.sp. ciceris, race 3. In potted experiment, four Trichoderma isolates were tested as seed treatment on three chickpea cultivars (JG-62 susceptible, Shasho moderately susceptible and JG-74 resistant) against F. oxysporum f.sp. ciceris, race 3. The result showed that T. harzianum and unidentified Trichoderma isolate T23 significantly reduced wilt severity and delayed disease onset. The degree of wilt severity and delay of disease onset varied with chickpea cultivars. Our study revealed that biological control agents such as Trichoderma can be a useful component of integrated chickpea Fusarium wilt management.

In Planta and Soil Quantification of Fusarium oxysporum f. sp. ciceris and Evaluation of Fusarium Wilt Resistance in Chickpea with a Newly Developed Quantitative Polymerase Chain Reaction Assay

Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris can be managed by risk assessment and use of resistant cultivars. A reliable method for the detection and quantification of F. oxysporum f. sp. ciceris in soil and chickpea tissues would contribute much to implementation of those disease management strategies. In this study, we developed a real-time quantitative polymerase chain reaction (q-PCR) protocol that allows quantifying F. oxysporum f. sp. ciceris DNA down to 1 pg in soil, as well as in the plant root and stem. Use of the q-PCR protocol allowed quantifying as low as 45 colony forming units of F. oxysporum f. sp. ciceris per gram of dry soil from a field plot infested with several races of the pathogen. Moreover, the q-PCR protocol clearly differentiated susceptible from resistant chickpea reactions to the pathogen at 15 days after sowing in artificially infested soil, as well as the degree of virulence between two F. oxysporum f. sp. ciceris races. Also, the protocol detected early asymptomatic root infections and distinguished significant differences in the level of resistance of 12 chickpea cultivars that grew in that same field plot infested with several races of the pathogen. Use of this protocol for fast, reliable, and cost-effective quantification of F. oxysporum f. sp. ciceris in asymptomatic chickpea tissues at early stages of the infection process can be of great value for chickpea breeders and for epidemiological studies in growth chambers, greenhouses and field-scale plots.

Biocontrol of Fusarium Wilt of Chickpea using Arbuscular Mycorrhizal Fungi andRhizobium leguminosorumBiovar

Abstract Present study deals with the biocontrol of Fusarium wilt of chickpea using arbuscular mycorrhizal fungi (AMF) Glomus hoi (Gh), Glomus fasciculatum (Gf) and Rhizobium leguminosorum Biovar. (Rl), which are the important members of rhizosphere and biological control agents, were examined on both the patho- system of Fusarium oxysporum f. sp. ciceris (Foc) and chickpea (Cicer arietinum). The colonization and nodulation of two biocontrol agents exhibited differences as a result of reciprocal interactions of these items as well as the effect of the Foc. Nodulation of Rl particularly decreased in triple inoculation. In addition, colonization of AMF significantly decreased in treatment of Foc+AMF than control, AMF. It was determined that single biological control agents inoculations were more effective than dual inoculations (AMF+Rl). When the morphological parameters of chickpea were considered, all of the morphological values were decreased in treatments which present Foc. Beside this all biological co...

Changes in the redox status of chickpea roots in response to infection by Fusarium oxysporum f. sp. ciceris: apoplastic antioxidant enzyme activities and expression of oxidative stress‐related genes

Activity levels of oxidative stress-related enzymes in the root apoplast during the interaction of WR315 (resistant) and JG62 (susceptible) chickpeas (Cicer arietinum L.) with the highly virulent race 5 of Fusarium oxysporum f. sp. ciceris were compared. Because this fungus develops asymptomatic infections in the chickpea root cortex in both susceptible and resistant plants, but only intrudes into the root xylem in the susceptible variety, the interactions were compared at three specific stages during disease development in JG62: (i) before symptom development (10 days after inoculation); (ii) at the time of appearance of the first disease symptoms (15-17 days after inoculation) and (iii) when all plants had developed disease symptoms (20-22 days after inoculation). Diamine oxidase (DAO), ascorbate peroxidase (APX), glutathione reductase (GR), guaiacol-dependent peroxidase and superoxide dismutase (SOD), but not catalase (CAT), were found in the apoplast of chickpea roots. In terms of APX activity, infection by the pathogen caused a different response in the incompatible compared to the compatible plant. In the case of GR, SOD and DAO activities, the pathogen caused the same response, but it developed earlier (i.e. GR and SOD) or to higher levels (i.e. DAO) in the incompatible interaction. Expression of apx, cat, sod, lipoxygenase (lox) and actin genes was also analysed in infected roots. Infection by F. oxysporum f. sp. ciceris race 5 only caused a significant change in the root expression of lox and actin genes. This up-regulation was earlier (lox) or higher (actin) in the incompatible than in the compatible interaction. Thus, changes in oxidative metabolism differ in compatible and incompatible interactions in Fusarium wilt of chickpea.

Influence of mineral amendment on disease suppressive activity of Pseudomonas fluorescens to Fusarium wilt of chickpea

Fusarium wilt caused by Fusarium oxysporum f. sp. ciceri causes considerable yield loss of chickpea. Pseudomonas fluorescens4-92 (Pf4-92) strain can suppress the disease. Amendment of zinc EDTA and copper EDTA could not suppress the disease significantly when used alone; however, they significantly suppressed the disease in presence of Pf4-92. In vitro observation showed that at 40, 30 and 20microgml(-1) concentrations of these minerals, i.e. Zn, Cu and Zn plus Cu, respectively, completely repressed the production of the phytotoxin, fusaric acid (FA). FA concentration (0.5microgml(-1)) has been shown to suppress the production of 2,4-diacetylphloroglucinol (DAPG) by Pf4-92, and DAPG, salicylic acid, pyochelin and pyoluteorin production was enhanced by these mineral amendments. In rockwool bioassays, Zn, Cu and Zn plus Cu amendments reduced FA production and enhanced DAPG production. This study demonstrates that Zn and Cu enhance biocontrol activity by reducing FA produced by the pathogen, F. oxysporum f. sp. ciceri.

Establishment of the Chickpea Wilt Pathogen Fusarium oxysporum f. sp. ciceris in the Soil through Seed Transmission

Chickpea wilt caused by Fusarium oxysporum f. sp. ciceris(FOC) is the most destructive disease in India. It is seed-borne as well as soil-borne pathogen. The role of seed-borne FOC in introducing and establishing wilt in FOC free soils is unknown. Using seeds of FOC infected chickpea cultivar K 850, we provided an evidence of establishing wilt disease in the FOC free soils within three crop cycles or seasons. In the first cycle, typical wilt symptoms were observed in 24 pots in 41 days after sowing. These 24 pots were used for second and third cycles without changing the soil. These 24 pots were sown with seeds collected from healthy plants of a susceptible cultivar JG 62, one seed per pot and development of wilt symptom was recorded. Wilt symptoms appeared in all the pots 26 days after sowing in second cycle and in 16 days after sowing in third cycle. On selective medium, all of the wilted plants yielded FOC in all the three cycles indicating that the mortality was due to wilt. FOC propagules on selective medium were 172, 1197, and 2280 <TEX>$g^{-1}$</TEX> soil at the end of the first, second, and third cycles, respectively. These studies indicated that Fusarium wilt of chickpea is seed-borne and seeds harvested from wilted plants when mixed with healthy seeds can carry the wilt fungus to new areas and can establish the disease in the soil to economic threshold levels within three seasons.

Fusarium wilt of chickpea: physiological specialization, genetics of resistance and resistance gene tagging

Chickpea wilt caused by Fusarium oxysporum f. sp. ciceris is one of the major yield limiting factors in chickpea. The disease causes 10–90% yield losses annually in chickpea. Eight physiological races of the pathogen (0, 1A, 1B/C, 2, 3, 4, 5 and 6) are reported so far whereas additional races are suspected from India. The distribution pattern of these races in different parts of the world indicates regional specificity for their occurrence leading to the perception that F. oxysporum f. sp. ciceris evolved independently in different regions. Pathogen isolates also exhibit differences in disease symptoms. Races 0 and 1B/C cause yellowing syndrome whereas 1A, 2, 3, 4, 5 and 6 lead to wilting syndrome. Genetics of resistance to two races (1B/C and 6) is yet to be determined, however, for other races resistance is governed either by monogenes or oligogenes. The individual genes of oligogenic resistance mechanism delay onset of disease symptoms, a phenomenon called as late wilting. Slow wilting, i.e., slow development of disease after onset of disease symptoms also occurs in reaction to pathogen; however, its genetics are not known. Mapping of wilt resistance genes in chickpea is difficult because of minimal polymorphism; however, it has been facilitated to great extent by the development of sequence tagged microsatellite site (STMS) markers that have revealed significant interspecific and intraspecific polymorphism. Markers linked to six genes governing resistance to six races (0, 1A, 2, 3, 4 and 5) of the pathogen have been identified and their position on chickpea linkage maps elucidated. These genes lie in two separate clusters on two different chickpea linkage groups. While the gene for resistance to race 0 is situated on LG 5 of Winter et al. (Theoretical and Applied Genetics 101:1155–1163, 2000) those governing resistance to races 1A, 2, 3, 4 and 5 spanned a region of 8.2 cM on LG 2. The cluster of five resistance genes was further subdivided into two sub clusters of 2.8 cM and 2.0 cM, respectively. Map-based cloning can be used to isolate the six genes mapped so far; however, the region containing these genes needs additional markers to facilitate their isolation. Cloning of wilt resistance genes is desirable to study their evolution, mechanisms of resistance and their exploitation in wilt resistance breeding and wilt management.

Genetic evidence for gametophytic selection of wilt resistant alleles in chickpea

Gametophytic selection can drastically reduce the number of selection cycles during crop improvement programs. The objective of the present investigation was to test whether the nature of inheritance of two unlinked disease-resistant loci, h(1) and h(2), against Fusarium wilt in chickpea (Cicer arietinum L.) under gametophytic (pollen) selection was similar to that already observed at sporophytic level. A homozygous dominant (H(1)H(1)H(2)H(2)) susceptible genotype JG-62 was crossed to a recessive (h(1)h(1)h(2)h(2)) resistant genotype WR-315 to produce 20 F(1) hybrid seeds. In the following generation, flower buds of 10 F(1) hybrid plants were subjected to toxin stress before anthesis and the remaining ten control F(1) plants' flowers were sprayed with water. Thirty-four selected BC(1) plants were generated by test crossing resistant WR-315 individuals with pollen from toxin-stressed F(1) individuals. Both control and treated F(1) plants were selfed to produce respective F(2) generations. Two DNA markers, CS-27(700bp) and A07C(430bp), linked to susceptible alleles H(1) and H(2), respectively, were used to study the inheritance patterns of h(1) and h(2) loci in the F(2) and BC(1) generations. One hundred and forty-four selected F(2), 129 control F(2), and 34 selected backcross individuals were tested for the presence or absence of DNA markers. Except for the control F(2), observed ratios of selected F(2) and BC(1) populations exhibited significant chi-square deviations from expected monogenic and digenic ratios. Our results suggest that gametophytic selection is as effective as that realized at the sporophytic level, and that the gametophytic selection can be an effective breeding tool for plant breeding programs.

Cell wall degrading enzymes in fusarium wilt of chickpea: correlation between pectinase and xylanase activities and disease development in plants infected with two pathogenic races of Fusarium oxysporum f. sp. ciceris

Production of cell wall degrading enzymes (CWDEs) polygalacturonase (PG), pectate lyase (PL), and xylanase was studied in chickpeas ( Cicer arietinum L. ‘P-2245’) inoculated with Fusarium oxysporum f. sp. ciceris (Padwick) Matuo &amp; K. Sato races 0 (mildly virulent, causing a yellowing syndrome) and 5 (highly virulent, causing a wilting syndrome) by the water-culture method. These CWDEs were similarly produced in both syndromes. PG and PL were the only enzymes occurring in roots and stems and attained the highest specific activity, this being generally higher for race 5 than for race 0. Gel filtration chromatography revealed a similar complement of in planta expressed pectinase isoforms, dominated by an endo-PG and two endo-PLs, the endo-PLs being differentially expressed by the two races. CWDE activities in roots and stems were positively correlated with development of yellowing and wilting. Exceptions to this were PG in stems, which was negatively correlated with the development of yellowing, and PG in roots, which showed a negative trend with development of either syndrome. The levels of CWDEs that significantly correlated with disease development were adequately described by exponential functions of disease progress. Results have implications for the role played by CWDEs in the early and later stages of pathogenesis in chickpea fusarium wilt.

Molecular characterization of Fusarium oxysporum f. sp. ciceri causing wilt of chickpea

Thirty isolates of Fusarium oxysporum f. sp. ciceri were isolated from rhizosphere soil of chickpea from different locations in Northern India. The amount of genetic variation was evaluated by polymerase chain reaction (PCR) amplification with a set of 40 RAPD primers and 2 IGS primers. Less than 10% of the amplified fragments in each case were polymorphic. Genetic similarity between each of the isolates was calculated and results indicate that there was little genetic variability among the isolates collected from the different locations. At the 0.75 similarity index the isolates divides into three groups. Isolates Foc-A18 , Foc-A19 , Foc-A20 forming a similar group and far different from other isolates. Key words: Fusarium oxysporum f. sp. ciceri , Fusarium wilt of chickpea, RAPD, ITS, IGS.

Temperature Response of Chickpea Cultivars to Races of Fusarium oxysporum f. sp. ciceris, Causal Agent of Fusarium Wilt.

Use of resistant cultivars and adjustment of sowing dates are important measures for management of Fusarium wilt in chickpeas (Cicer arietinum). In this study, we examined the effect of temperature on resistance of chickpea cultivars to Fusarium wilt caused by various races of Fusarium oxysporum f. sp. ciceris. Greenhouse experiments indicated that the chickpea cultivar Ayala was moderately resistant to F. oxysporum f. sp. ciceris when inoculated plants were maintained at a day/night temperature regime of 24/21°C but was highly susceptible to the pathogen at 27/25°C. Field experiments in Israel over three consecutive years indicated that the high level of resistance of Ayala to Fusarium wilt when sown in mid- to late January differed from a moderately susceptible reaction under warmer temperatures when sowing was delayed to late February or early March. Experiments in growth chambers showed that a temperature increase of 3°C from 24 to 27°C was sufficient for the resistance reaction of cultivars Ayala and PV-1 to race 1A of the pathogen to shift from moderately or highly resistant at constant 24°C to highly susceptible at 27°C. A similar but less pronounced effect was found when Ayala plants were inoculated with F. oxysporum f. sp. ciceris race 6. Conversely, the reaction of cultivar JG-62 to races 1A and 6 was not influenced by temperature, but less disease developed on JG-62 plants inoculated with a variant of race 5 of F. oxysporum f. sp. ciceris at 27°C compared with plants inoculated at 24°C. These results indicate the importance of appropriate adjustment of temperature in tests for characterizing the resistance reactions of chickpea cultivars to the pathogen, as well as when determining the races of isolates of F. oxysporum f. sp. ciceris. Results from this study may influence choice of sowing date and use of chickpea cultivars for management of Fusarium wilt of chickpea.

IDENTIFICATION OF RHIZOBIUM ISOLATES POSSESSING ANTAGONISTIC ACTIVITY AGAINST FUSARIUM OXYSPORUM F.SP. CICERIS, THE CAUSAL AGENT OF FUSARIUM WILT OF CHICKPEA

Using two cultivars (the susceptible ILC482 and the moderately resistant INRAT 87/1) of chickpea (Cicer arietinum), the antagonistic activities of 21 Rhizobium isolates were tested in vitro in dual culture, and in vivo under greenhouse and field conditions against Fusarium oxysporum f.sp. ciceris (Foc) race 0, the causal agent of Fusarium wilt of chickpea. In dual culture, 14 isolates inhibited the mycelial growth of the pathogen more than 30% and the most effective were Rh8, Rh11, Rh16 and PchSOM, which inhibited fungal growth more than 50%. Among the 14 Rhizobium isolates tested for volatiles, cyanide production and phosphate solubilisation, 8 significantly inhibited fungal growth by producing volatiles, 6 were positive for cyanide production and only three were able to solubilise phosphate. Isolate Rh8 produced the highest levels of volatiles, giving more than 10.7% fungal inhibition, and was the only one positive for both cyanide production and phosphate solubilisation. Greenhouse experiments on the same 14 isolates revealed the effectiveness of five: PchDMS, Pch 121, Rh5, Rh17 and Pch43. These reduced the percentage of wilted plants in both susceptible and moderately resistant cultivars. These percentages ranged from 12.5 to 54.6% in the susceptible cultivar ILC482 and from 8.3 to 29.1% in the moderately resistant cultivar INRAT 87/1. The best disease control was achieved by isolate PchDMS. Despite its effectiveness in vitro, isolate Rh8 was ineffective under greenhouse conditions. Field experiments showed that none of the 14 Rhizobium isolates significantly reduced the percentage of wilted plants of the susceptible cultivar ILC482, although with the moderately resistant cultivar INRAT 87/1 eight of the isolates significantly reduced wilt incidence. Inoculation of seeds with these isolates reduced the percentage of diseased plants from more than 48.6% in infected control plants to less than 20.3% in plants inoculated with the bacteria and infected with the pathogen. The best protection against disease was obtained with isolates Pch43 and Rh4, which reduced the percentage of wilted plants to less than 8%. Besides their beneficial effects on disease control, our studies showed that rhizobia may improve plant growth and yield. These results indicate that Rhizobium isolates could be effective under commercial conditions in reducing the deleterious effects of Fusarium wilt.

Extracellular xylanases from two pathogenic races of Fusarium oxysporum f. sp. ciceris: enzyme production in culture and purification and characterization of a major isoform as an alkaline endo-β-(1,4)-xylanase of low molecular weight

Fusarium oxysporum f. sp. ciceris, the causal agent of Fusarium wilt of chickpea, comprises eight pathogenic races and two pathotypes. Races 0 and 5, representative of the least virulent yellowing pathotype and the most virulent wilt pathotype, respectively, produced extracellular xylanases when grown on minimal medium supplemented with either 1% commercial birchwood xylan or 0.3% chickpea cell walls. The pattern of extracellular proteins analysed by denaturing polyacrylamide gel electrophoresis in the two media presented some minor but distinctive differences between fungal races. By preparative isoelectrofocusing, the xylanase activity in cell wall-culture filtrates could be resolved into basic and neutral fractions with pI values around to 10 and 8, respectively, whereas the xylan-culture filtrates contained an additional acidic fraction of pI around 4. A common major xylanase was purified 7-fold to homogeneity by cation-exchange chromatography and chromatofocusing. The purified xylanase has a molecular weight of 21.6 kDa, optimum pH and temperature of 5.5 and 55 degrees C, respectively, pI in the range of 8.2 to 9.0, and Km and Vmax values of 2.24 mg ml(-1) (birchwood xylan as substrate) and 1200 nkat mg(-1) protein (72 U mg(-1) protein), respectively. The enzyme has an endo mode of action, hydrolysing xylan to xylobiose and higher short-chain xylooligosaccharides without forming free xylose.

Effect of Iron Availability on Induction of Systemic Resistance to Fusarium Wilt of Chickpea by Pseudomonas spp.

Selected isolates of Pseudomonas fluorescens (Pf4-92 and PfRsC5) and P aeruginosa (PaRsG18 and PaRsG27) were examined for growth promotion and induced systemic resistance against Fusarium wilt of chickpea. Significant increase in plant height was observed in Pseudomonas treated plants. However, plant growth was inhibited when isolates of Pseudomonas were used in combination with Fusarium oxysporum f. sp. ciceri (FocRsl). It was also observed that the Pseudomonas spp. was colonized in root of chickpea and significantly suppressed the disease in greenhouse condition. Rock wool bioassay technique was used to study the effect of iron availability on the induction of systemic resistance to Fusarium wilt of chickpea mediated by the Pseudomonas spp. All the isolates of Pseudomonas spp. showed greater disease control in the induced systemic resistance (ISR) bioassay when iron availability in the nutrient solution was low. High performance liquid chromatography (HPLC) analysis indicated that all the bacterial isolates produced more salicylic acid (SA) at low iron (10 μM EDDHA) than high iron availability (10 μFe3+ EDDHA). Except PaRsG27, all the three isolates produced more pseudobactin at low iron than high iron availability.

Integrated Management of Fusarium Wilt of Chickpea with Sowing Date, Host Resistance, and Biological Control

ABSTRACT A 3-year experiment was conducted in field microplots infested with Fusarium oxysporum f. sp. ciceris race 5 at Córdoba, Spain, in order to assess efficacy of an integrated management strategy for Fusarium wilt of chickpea that combined the choice of sowing date, use of partially resistant chickpea genotypes, and seed and soil treatments with biocontrol agents Bacillus megaterium RGAF 51, B. subtilis GB03, nonpathogenic F. oxysporum Fo 90105, and Pseudomonas fluorescens RG 26. Advancing the sowing date from early spring to winter significantly delayed disease onset, reduced the final disease intensity (amount of disease in a microplot that combines disease incidence and severity, expressed as a percentage of the maximum possible amount of disease in that microplot), and increased chickpea seed yield. A significant linear relationship was found between disease development over time and weather variables at the experimental site, with epidemics developing earlier and faster as mean temperature increased and accumulated rainfall decreased. Under conditions highly conducive for Fusarium wilt development, the degree of disease control depended primarily on choice of sowing date, and to a lesser extent on level of resistance of chickpea genotypes to F. oxysporum f. sp. ciceris race 5, and the biocontrol treatments. The main effects of sowing date, partially resistant genotypes, and biocontrol agents were a reduction in the rate of epidemic development over time, a reduction of disease intensity, and an increase in chickpea seedling emergence, respectively. Chickpea seed yield was influenced by all three factors in the study. The increase in chickpea seed yield was the most consistent effect of the biocontrol agents. However, that effect was primarily influenced by sowing date, which also determined disease development. Effectiveness of biocontrol treatments in disease management was lowest in January sowings, which were least favorable for Fusarium wilt. Sowing in February, which was moderately favorable for wilt development, resulted in the greatest increase in seed yield by the biocontrol agents. In March sowings, which were most conducive for the disease, the biocontrol agents delayed disease onset and increased seedling emergence. B. subtilis GB03 and P. fluorescens RG 26, applied either alone or each in combination with nonpathogenic F. oxysporum Fo 90105, were the most effective treatments at suppressing Fusarium wilt, or delaying disease onset and increasing seed yield, respectively. The importance of integrating existing control practices, partially effective by themselves, with other control measures to achieve appropriate management of Fusarium wilt and increase of seed yield in chickpea in Mediterranean-type environments is demonstrated by the results of this study.

Influence of temperature on plant–rhizobacteria interactions related to biocontrol potential for suppression of fusarium wilt of chickpea

Seed and soil treatment with Pseudomonas fluorescens RGAF 19, P. fluorescens RG 26, Bacillus megaterium RGAF 51 and Paenibacillus macerans RGAF 101 can suppress fusarium wilt of chickpea (Cicer arietinum), but the extent of disease suppression by these rhizobacteria is modulated by soil temperature. In this work, the effect of temperature on plant–rhizobacteria interactions was assessed in relation to biocontrol potential for suppression of fusarium wilt of chickpea. Seed and soil treatment with those rhizobacteria delayed seedling emergence compared with nontreated controls, and either increased or had no deleterious effect on chickpea growth. Pseudomonas fluorescens isolates significantly increased chickpea shoot dry weight at 20°C and root dry weight at 25 and 30°C. All bacterial isolates colonized the chickpea rhizosphere and internal stem tissues at 20, 25 and 30°C, and there was a positive linear trend between bacterial population size in the rhizosphere and temperature increase. The maximum inhibition of mycelial growth and conidial germination of Fusarium oxysporum f. sp. ciceris race 5 in vitro occurred at a temperature range optimal for bacterial growth and production of inhibitory metabolites. These results demonstrate the need to understand the effects of environmental factors on the biological activities of introduced rhizobacteria of significant importance for plant disease suppression.

Induction of Defense Related Enzymes and Pathogenesis Related Proteins in Pseudomonas fluorescens-Treated Chickpea in Response to Infection by Fusarium oxysporum f. sp. ciceri

Pseudomonas fluorescens 1–94 induced systemic resistance in chickpea against Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceri by the synthesis and accumulation of phenolic compounds, phenylalanine ammonia lyase (PAL) and pathogenesis related (PR) proteins (chitinase, β-1,3-glucanase and peroxidase). Time-course accumulation of these enzymes in chickpea plants inoculated with P. fluorescens was significantly (LSD, P =0.05) higher than control. Maximum activities of PR-proteins were recorded at 3 days after inoculation in all induced plants; thereafter, the activity decreased progressively. Five PR peroxidases detected in induced chickpea plants. Molecular mass of these purified peroxidases was 20, 29, 43, 66 and 97 kDa. Purified peroxidases showed antifungal activity against plant pathogenic fungi.