Virulence Analysis and Oligonucleotide Fingerprinting to Detect Diversity Among Indian Isolates of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">ciceris</Emphasis> Causing Chickpea Wilt

Virulence analysis of 64 isolates of Fusarium oxysporum f. sp. ciceris causing chickpea wilt collected from major chickpea growing states of India on 14 varieties, including 10 international differentials revealed that the isolates from each state were highly variable. Based on the reactions on international differentials, more than one race was found to be prevalent in every state. Majority of the isolates were not matched with the race specific reactions. Therefore, some of the cultivars, namely, GPF 2, DCP 92-3, and KWR 108 should be included as new differentials to obtain clear-cut differential responses. Randomly amplified polymorphic DNA (RAPD), inter-simple sequence repeat (ISSR), and simple sequence repeat (SSR) markers were used to assess the genetic diversity of these isolates. Unweighted paired group method with arithmetic average (UPGMA) cluster analysis was used to divide the isolates into distinct clusters. The clusters generated by RAPD grouped all isolates into three categories at 25% genetic similarity and into two major categories at 30% genetic similarity. ISSR and SSR analyses also grouped all the isolates into two major categories. Majority of the isolates from Punjab and a few from Rajasthan were grouped in one category while the isolates from all other states were grouped in another suggesting the existence of diverse genetic populations of the pathogen at the same location. Some of the RAPD (OPM 6, OPI 9, P 17, OPN 4, OPF 1, P 17, P 21, and SC 1), ISSR (ISSR 7, ISSR 11, and ISSR 12) and SSR (MB 17) markers clearly distinguished area specific isolates.     

Combined application of botanical formulations and biocontrol agents for the management of Fusarium oxysporum f. sp. cubense (Foc) causing Fusarium wilt in banana

Plant products along with biocontrol agents were tested against Fusarium wilt of banana caused by Fusarium oxysporum f. sp. cubense (Foc). Of the 22 plant species tested, the leaf extract of Datura metel (10%) showed complete inhibition of the mycelial growth of Foc. Two botanical fungicides, Wanis 20 EC and Damet 50 EC along with selected PGPR strains with known biocontrol activity, Pseudomonas fluorescens 1, Pf1 and Bacillus subtilis, TRC 54 were tested individually and in combination for the management of Fusarium wilt under greenhouse and field conditions. Combined application of botanical formulation and biocontrol agents (Wanis 20 EC + Pf1 + TRC 54) reduced the wilt incidence significantly under greenhouse (64%) and field conditions (75%). Reduction in disease incidence was positively correlated with the induction of defense-related enzymes peroxidase (PO) and polyphenol oxidase (PPO). Three antifungal compounds (two glycosides and one ester) in D. metel were separated and identified using TLC, RP-HPLC (Reverse Phase-High Pressure Liquid Chromatography) and mass spectrometry. In this study it is clear that combined application of botanical formulations and biocontrol agents can be very effective in the management of Fusarium wilt of banana.     

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.     

Induction of systemic resistance in chickpea against Fusarium wilt by Bacillus strains

Plant growth promoting rhizobacteria (PGPR) strains Rb29 (B. amyloliquefaciens MF352007), Bs1 (B. subtilis MF352017) and Bt1 (B. tequilensis MF352019) were tested for growth promotion and for their ability to induce systemic resistance against Fusarium wilt, a vascular disease of chickpea, using two methods that include whole plant and a split-root system. Bacillus strains and Fusarium oxysporum f. sp. ciceris (FOC) were inoculated on separate halves of roots of chickpea seedlings at the same time and then planted in separate pots either in superposition or one side of the other. All Bacillus strains systemically induced resistance against FOC, and significantly (p < 0.05) reduced the wilt disease by 98–100%. Application of Bacillus strains effectively enhanced plant growth, leading to increased plant height, root length, a fresh and dry weight of shoots and roots. These results help to explain the role of strains of Bacillus in growth promotion and biological control of Fusarium wilt in chickpea. This is the first report of systemic-induced resistance against Fusarium wilt in chickpea obtained by application of Bacillus strains to a root system spatially separated from the FOC-inoculated root.     

Biological control ofFusarium oxysporum f.sp.cubense in banana by inoculation withPseudomonas fluorescens

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.     

Evaluation of Streptomyces spp. and Bacillus spp. for biocontrol of Fusarium wilt in chickpea (Cicer arietinum L.)

A study was carried out to test direct and indirect antagonistic effect against Fusarium wilt, caused by Fusarium oxysporum f. sp. ciceri (FOC), and plant growth-promoting (PGP) traits of bacteria isolated from rhizosphere soils of chickpea (Cicer arietinum L.). A total of 40 bacterial isolates were tested for their antagonistic activity against FOC and of which 10 were found to have strong antagonistic potential. These were found to be Streptomyces spp. (five isolates) and Bacillus spp. (five isolates) in the morphological and biochemical characterisation and 16S rDNA analysis. Under both greenhouse and wilt sick field conditions, the selected Streptomyces and Bacillus isolates reduced disease incidence and delayed expression of symptoms of disease, over the non-inoculated control. The PGP ability of the isolates such as nodule number, nodule weight, shoot weight, root weight, grain yield and stover yield were also demonstrated under greenhouse and field conditions over the non-inoculated control. Among the ten isolates, Streptomyces sp. AC-19 and Bacillus sp. BS-20 were found to have more potential for biocontrol of FOC and PGP in chickpea. This investigation indicates that the selected Streptomyces and Bacillus isolates have the potential to control Fusarium wilt disease and to promote plant growth in chickpea.     

Combinatorial effect of endophytic and plant growth promoting rhizobacteria against wilt disease of Capsicum annum L. caused by Fusarium solani

Combination of biocontrol agents that are compatible with each other is a strategic approach to control the plant disease and pest. The present study was designed to evaluate the protective effects of compatible endophytic bacterial strains (Bacillus subtilis; EPCO16 and EPC5) and rhizobacterial strain (Pseudomonas fluorescens; Pf1) against chilli wilt disease caused by Fusarium solani. Our results showed that B. subtilis (EPCO16 and EPC5) and P. fluorescens (Pf1) were compatible and effectively inhibited the growth of the F. solani. The application of endophytic and rhizobacterial strains, singly and in combination in green house and field conditions were found to be effective in controlling the chilli Fusarium wilt disease by inducing systemic resistance (ISR) as evidenced by enhanced activities of PO, PPO, PAL, β-1,3-glucanase, Chitinase and Phenolic involved in the synthesis of phytolaexins thereby promoting the growth of plants. However, combinations of EPCO16 + EPC5 + Pf1 bacterial strains were more effective than single agents. These findings suggest that synergistic interactions of biocontrol agents may be responsible for the management of chilli wilt disease caused by F. solani.     

Salicylic acid and salicylic acid sensitive and insensitive catalases in different genotypes of chickpea against Fusarium oxysporum f. sp. ciceri

Differential expression of catalase isozymes in different genotypes of chickpea resistant genotypes- A1, JG-315, JG-11, WR-315, R₁-315, Vijaya, ICCV-15017, GBS-964, GBM-10, and susceptible genotypes- JG-62, MNK, ICCV-08321, ICCV-08311, KW-104, ICCV-08123, ICC-4951, ICC-11322, ICC-08116 for wilt disease caused by Fusarium oxysporum. f. sp. ciceri (Foc) was analyzed. Salicylic acid (SA) and H₂O₂ concentrations were determined in control as well as in plants infected with F. ciceri and found that the high and low levels of salicylic acid and H₂O₂ in resistant and susceptible genotypes of chickpea respectively. Catalase isozyme activities were detected in the gel and found that no induction of new catalases was observed in all the resistant genotypes and their some of the native catalase isozymes were inhibited; whereas, induction of multiple catalase isozymes was observed in all the screened susceptible genotypes and their activities were not inhibited upon Foc or SA treatments. The above results support the possible role of these isozymes as a marker to identify which genotype of chickpea is expressing systemic acquired resistance.     

Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen

Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus. Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R. solani in vitro. Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P. fluorescens strains was evaluated. The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2. A significant relationship between the antagonistic potential of P. fluorescens against R. solani and its level of beta-1,3-glucanase, SA and HCN was observed.     

Molecular characterization of Macrophomina phaseolina and Fusarium species by a single primer RAPD technique

Charcoal root rot and wilt, are two economically important diseases of many crop plants in North and South America, Asia and Africa and some parts of Europe. Genetic variation in 43 isolates of Macrophomina phaseolina and 22 isolates of Fusarium species, collected from geographically distinct regions over a range of hosts, was studied using random amplified polymorphic DNA (RAPD) markers. Initially, 210 arbitrary nucleotide (10-mer) primers were tested for amplification of genomic DNA of one M. phaseolina isolate, 70 primers amplified the genomic DNA of M. phaseolina. One primer OPA-13 (5'-CAGCACCCAC-3') produced fingerprint profiles, which clearly distinguished between the different isolates of M. phaseolina. UPGMA analysis classified these isolates into five major groups. By primer OPA-13, 22 isolates of pathogenic and non-pathogenic Fusarium species of different formae-speciales and races, were also distinguished from M. phaseolina. This marker is useful for distinguishing between these two important plant pathogens irrespective of hosts, virulence spectrum and races. This is the first report of reliable diagnosis of two soilborne pathogens (root/collar rot and wilt causing pathogens) at the level of isolates, formae-speciales and races by a single primer RAPD procedure with uniform PCR conditions.     

Molecular mapping of wilt resistance genes in chickpea

Fusarium wilt is a widespread and serious chickpea disease caused by the soil-borne fungus Fusarium oxysporum f.sp. ciceri (Foc). We evaluated an F₉ recombinant inbred line population of chickpea for resistance to three Foc races (1, 2 and 3) in pot culture experiments and identified flanking and tightly linked DNA markers for the resistance genes. The simple sequence repeat markers H3A12 and TA110 flanked the Foc1 locus at 3.9 and 2.1 cM, respectively, while Foc2 was mapped 0.2 cM from TA96 and 2.7 cM from H3A12. The H1B06y and TA194 markers flanked the Foc3 locus at 0.2 and 0.7 cM, respectively. These markers were also validated using 16 diverse chickpea genotypes. Identification of tightly linked flanking markers for wilt resistance genes will be useful for their exploitation in breeding programs and to understand the mechanism of resistance and evolution of the genes. S. J. M. Gowda and P. Radhika contributed equally to this study.     

Suppression of maize root diseases caused by Macrophomina phaseolina, Fusarium moniliforme and Fusarium graminearum by plant growth promoting rhizobacteria

A plant growth-promoting isolate of a fluorescent Pseudomonas sp. EM85 and two bacilli isolates MR-11(2) and MRF, isolated from maize rhizosphere, were found strongly antagonistic to Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina, causal agents of foot rots and wilting, collar rots/stalk rots and root rots and wilting, and charcoal rots of maize, respectively. Pseudomonas sp. EM85 produced antifungal antibiotics (Afa⁺), siderophore (Sid⁺), HCN (HCN⁺) and fluorescent pigments (Flu⁺) besides exhibiting plant growth promoting traits like nitrogen fixation, phosphate solubilization, and production of organic acids and IAA. While MR-11(2) produced siderophore (Sid⁺), antibiotics (Afa⁺) and antifungal volatiles (Afv⁺), MRF exhibited the production of antifungal antibiotics (Afa⁺) and siderophores (Sid⁺). Bacillus spp. MRF was also found to produce organic acids and IAA, solubilized tri-calcium phosphate and fixed nitrogen from the atmosphere. All three isolates suppressed the diseases caused by Fusarium moniliforme, Fusarium graminearum and Macrophomina phaseolina in vitro. A Tn5:: lac Z induced isogenic mutant of the fluorescent Pseudomonas EM85, M23, along with the two bacilli were evaluated for in situ disease suppression of maize. Results indicated that combined application of the two bacilli significantly (P = 0.05) reduced the Macrophomina-induced charcoal rots of maize by 56.04%. Treatments with the MRF isolate of Bacillus spp. and Tn5:: lac Z mutant (M23) of fluorescent Pseudomonas sp. EM85 significantly reduced collar rots, root and foot rots, and wilting of maize caused by Fusarium moniliforme and F. graminearum (P = 0.05) compared to all other treatments. All these isolates were found very efficient in colonizing the rhizotic zones of maize after inoculation. Evaluation of the population dynamics of the fluorescent Pseudomonas sp. EM85 using the Tn5:: lac Z marker and of the Bacillus spp. MRF and MR-11(2) using an antibiotic resistance marker revealed that all the three isolates could proliferate successfully in the rhizosphere, rhizoplane and endorhizosphere of maize, both at 30 and 60 days after seeding. Four antifungal compounds from fluorescent Pseudomonas sp. EM85, one from Bacillus sp. MR-11(2) and three from Bacillus sp. MRF were isolated, purified and tested in vitro and in thin layer chromatography bioassays. All these compounds inhibited R. solani, M. phaseolina, F. moniliforme, F. graminearum and F. solani strongly. Results indicated that antifungal antibiotics and/or fluorescent pigment of fluorescent Pseudomonas sp. EM85, and antifungal antibiotics of the bacilli along with the successful colonization of all the isolates might be involved in the biological suppression of the maize root diseases.     

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.     

Isolation of Endophytic Actinomycetes From Roots and Leaves of Banana (Musa Acuminata) Plants and Their Activities Against Fusarium oxysporumf. sp. cubense

Two hundred and forty-two actinomycete strains were isolated from the interior of leaves and roots of healthy and wilting banana plants. Most of them were streptomycetes, Streptomyces griseorubiginosus-like strains were the most frequently isolated strains. Community analysis demonstrated increased actinomycete diversity in wilting leaves compared to that in healthy leaves, similar actinomycete communities were found in wilting and healthy roots. Screening of the isolates for antagonistic activity against Fusarium oxysporumf. sp. cubenserevealed that the proportion of antagonistic streptomycetes in healthy roots was higher than that in wilting roots (P < 0.01), but no difference was found between antagonistic strains isolated from healthy and wilting leaves. The potential biological control of Panama disease of banana by endophytic streptomycetes, especially Streptomyces griseorubiginosus-like strains was discussed.     

Utilization of chemical inducers of resistance and Cryptococcus flavescens OH 182.9 to reduce Fusarium head blight under greenhouse conditions

Four chemicals [salicylic acid (SA), sodium salt of salicylic acid (NaSA), isonicotinic acid (INA), and DL-β-amino-n-butyric acid (BABA)] and the yeast antagonist Cryptococcus flavescens (=C. nodaensis nomen nudum) OH 182.9 were evaluated separately or together for the ability to reduce Fusarium head blight (FHB) of wheat in the greenhouse. When sprayed onto wheat heads at 3 days prior to pathogen challenge with Gibberella zeae, NaSA and INA at 10 mM significantly reduced FHB severity compared to the non-treated disease control. Applied at concentrations of 1 and 5 mM at 3 days before pathogen challenge, NaSA or INA in combination with OH 182.9 did not significantly reduce FHB severity compared to either treatment alone, though the lowest disease severity values frequently were associated with the combination treatments. When sprayed onto wheat heads just beginning to emerge from boot at 10 days prior to pathogen inoculation, NaSA, INA, and BABA at 1 mM significantly reduced FHB severity indicating that induced systemic resistance was at least partially responsible for the reduction of FHB disease. Induced FHB resistance was achieved by treating wheat with INA at concentrations as low as 0.1 mM. In only one instance was 100-kernel weight affected by any chemical or combination of chemicals with OH 182.9 treatment. Data from our studies in the greenhouse suggest that chemical inducers can induce resistance in wheat against FHB, and that further efforts are warranted to explore the potential of improved control of FHB disease by incorporating chemical inducers with the FHB biocontrol agent OH 182.9.     

Interaction of Fusarium oxysporum f. sp. ciceri and Meloidogyne javanica on Cicer arietinum

Interaction of Meloidogyne javanica and Fusarium oxysporum f. sp. ciceri was studied on Fusarium wilt-susceptible (JG 62 and K 850) and resistant (JG 74 and Avrodhi) chickpea cultivars. In greenhouse experiments, inoculation of M. javanica juveniles prior to F. oxysporum f. sp. ciceri caused greater wilt incidence in susceptible cultivars and induced vascular discoloration in roots of resistant cultivars. Nematode reproduction was greatest (P = 0.05) at 25 °C. Number of galls and percentage of root area galled increased when the temperature was increased from 15 °C to 25 °C. Wilt incidence was greater at 20 °C than at 25 °C. Chlorosis of leaves and vascular discoloration of plants did not occur at 15 °C. The nematode enhanced the wilt incidence in wilt-susceptible cultivars only at 25 °C. Interaction between the two pathogens on shoot and root weights was significant only at 20 °C, and F. o. ciceri suppressed the nematode density at this temperature. Wilt incidence was greater in clayey (48% clay) than in loamy sand (85% sand) soils. The nematode caused greater plant damage on loamy sand than on clayey soil. Fusarium wilt resistance in Avrodhi and JG 74 was stable in the presence of M. javanica across temperatures and soil types.     

Effects of Commercial and Indigenous Microorganisms on Fusarium Wilt Development in Chickpea

The purpose of this research was to determine whetherBacillus subtilis,nonpathogenicFusarium oxysporum,and/orTrichoderma harzianum,applied alone or in combination to chickpea (Cicer arietinumL.) cultivars ‘ICCV 4’ and ‘PV 61’ differing in their levels of resistance to Fusarium wilt, could effectively suppress disease caused by the highly virulent race 5 ofFusarium oxysporumf. sp.ciceris.Seeds of both cultivars were sown in soil amended with the three microbial antagonists, alone or in combination, and 7 days later seedlings were transplanted into soil infested with the pathogen. All three antagonistic microorganisms effectively colonized the roots of both chickpea cultivars, whether alone or in combination, and significantly suppressed Fusarium wilt development. In comparison with the control, the incubation period for the disease was delayed on average about 3 days and the final disease severity index and standardized area under the disease progress curve were reduced significantly between 14 and 33% and 16 and 42%, respectively, by all three microbial antagonists. Final disease incidence only was reduced byB. subtilis(18–25%) or nonpathogenicF. oxysporum(18%). The extent of disease suppression was higher and more consistent in ‘PV 61’ than in ‘ICCV 4’ whether colonized byB. subtilis,nonpathogenicF. oxysporum,orT. harzianum.The combination ofB. subtilis+T. harzianumwas effective in suppressing Fusarium wilt development but it did not differ significantly from treatments with either of these antagonists alone. In contrast, the combination ofB. subtilis+ nonpathogenicF. oxysporumtreatment was not effective but either antagonist alone significantly reduced disease development.     

Organic matter fractions by SP-MAS C NMR and microbial communities involved in the suppression of Fusarium wilt in organic growth media

Fusarium wilt is an economically important disease in carnation and tomato plants. The use of suppressive plant growth media has become an alternative method for plant disease control due to the lack of effective chemical control measures. Plant disease suppressiveness is sustained only in plant growth media with an adequate organic matter (OM) composition. Carbohydrate polymers are the most important sources of carbon nutrient for microbial community in these media, mainly consisting of cellulose and hemicellulose. This determines microbial activity, biomass and selects microbial communities in plant growth media, which are reported factors associated with Fusarium wilt suppressiveness.This work determined OM carbon functional groups using Single Pulse Magic Angle Spinning ¹³C-Nuclear Magnetic Resonance (SP-MAS ¹³C-NMR) in three plant growth media with different suppressiveness levels to Fusarium wilt in two crops, carnation and tomato. We propose that the critical role of OM to sustain naturally occurring suppressiveness in those media is not related with cellulose reserve. This could be explained because cellulose protected by lignin encrustation is not available to microbial degradation, meaning that cellulose availability is critical to sustenance of microorganism-mediated biological control. However, the hemicellulose relative abundance (peak 175 ppm) was associated to Fusarium wilt suppression level in plant growth media studied.Carbon source availability in OM was related to microbial biomass and econutritional group population densities involved in biocontrol. For these composts, Bacillus spp., oligotrophic and cellulolytic actinomycetes, and oligotrophic actinomycetes/oligotrophic bacteria and cellulolytic actinomycetes/cellulolytic bacteria ratios were indicated as microbial populations potentially involved in suppression.     

耐热木霉菌株筛选及其对热作区香蕉促生效应的研究

[目的]为筛选安全、高效的耐热木霉功能菌株,有效促进热作区香蕉的生产,本研究从热作区土壤分离筛选适温范围较宽的木霉菌株,研制木霉生物有机肥,并研究其对香蕉枯萎病发病率、果实产量及品质的影响.[方法]通过稀释涂布法筛选出木霉菌株,根据菌株在不同温度下的生长情况、拮抗尖孢菌能力及酶活强弱进行菌株复筛;将复筛所得菌株试制成生...