In vitro biocontrol activity of halotolerant Streptomyces aureofaciens K20: A potent antagonist against Macrophomina phaseolina (Tassi) Goid

A halotolerant actinobacterial strain isolated from salinity affected soil of Eastern Indo-Gangetic plains (IGP), Uttar Pradesh, India, was characterised for its antagonistic potential against Macrophomina phaseolina by dual-culture assay. It was shown to effectively inhibit the growth of M. phaseolina with an inhibition zone of 27 ± 1.33 mm. Further the actinobacterial strain was evaluated for its plant growth promoting (PGP) properties and its ability to produce biocontrol related extracellular enzymes viz. amylase, protease, cellulase, chitinase, gelatinase and urease. The results revealed that the actinobacterial strain had PGP potential along with positive assay for amylase, chitinase and urease. The interaction study between antagonist strain and fungal pathogen, performed by scanning electron microscopy technique revealed that the actinobacterium was able to damage fungal mycelia may be due to chitinase, establishing its role as a potential antagonist against M. phaseolina. The actinobacterial isolate was characterised by 16S rDNA gene sequencing, and was identified as Streptomyces genera. The identified gene sequence was deposited to NCBI GenBank with an accession number KP331758.     

Microsatellites from the charcoal rot fungus (Macrophomina phaseolina)

Microsatellite loci were identified from the charcoal rot fungus (Macrophomina phaseolina). Primer pairs for 46 loci were developed, and of these, 13 were optimized and screened using genomic DNA from 55 fungal isolates collected predominantly from two soybean fields in Mississippi. Twelve of the optimized loci were polymorphic and the number of alleles per locus ranged from 6 to 22. These microsatellites will be useful in population and pathogenicity studies to correspond with development of potential disease-resistant soybean and other susceptible crops.     

Plant defense approach of Bacillus subtilis (BERA 71) against Macrophomina phaseolina (Tassi) Goid in mung bean

This study was aimed to elucidate the mitigation mechanism of an endophytic bacterium, Bacillus subtilis (BERA 71) against Macrophomina phaseolina (Tassi) Goid disease in mung bean. M. phaseolina reduced the plant growth by inducing disease, hydrogen peroxide (H₂O₂) and lipid peroxidation. The inoculation of B. subtilis to diseased plants increased chlorophyll, ascorbic acids, and superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and glutathione reductase activities, and while inhibited H₂O₂ and lipid peroxidation for enhancing plant growth. In addition, B. subtilis association in plants mitigated the M. phaseolina infection due to increase of indole acetic acids and indole butyric acid, and also a decrease of abscisic acid. However, the nutrients (N, K, Ca, Mg, Zn, Cu, Mn and Fe) were increased, except Na in M. phaseolina diseased plants treated with B. subtilis. The result of this study suggests that B. subtilis interaction with plants can modulate the metabolism of pigments, hormones, antioxidants and nutrients against M. phaseolina to induce disease resistance in mung bean.     

Two pathogenesis-related peroxidases in greengram (Vigna radiata (L.) wilczek) leaves and cultured cells induced by Macrophomina phaseolina (Tassi) Goid. and its elicitor

An elicitor has been isolated from Macrophomina phaseolina, the root rot and leaf blight pathogen of greengram. Suspension-cultured cells of greengram were established which responded to the fungal elicitor. When greengram leaves were inoculated with M. phaseolina two new peroxidases appeared. Similarly, two new peroxidases could be detected in suspension-cultured greengram cells when treated with the fungal elicitor. These peroxidases were purified by column chromatography and their molecular masses were 27 and 38 kDa. The new peroxidases detected in both leaves and cultured cells appear to be similar with the same molecular weights.     

Evaluation of bacteria isolated from rice rhizosphere for biological control of charcoal rot of sorghum caused by <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> (Tassi) Goid.

A total of 360 bacteria, isolated from the rhizospheres of a system of rice intensification (SRI) fields, were characterized for the production of siderophore, fluorescence, indole acetic acid (IAA), hydrocyanic acid (HCN) and solubilization of phosphorus. Of them, seven most promising isolates (SRI-156, -158, -178, -211, -229, -305 and -360) were screened for their antagonistic potential against Macrophomina phaseolina (causes charcoal rot in sorghum) by dual culture assay, blotter paper assay and in greenhouse. All the seven isolates inhibited M. phaseolina in dual culture assay, whereas six isolates solubilized phosphorous (except SRI-360), all seven produced siderophore, four produced fluorescence (except SRI-178, -229 and -305), six produced IAA (except SRI-305) and five produced HCN (except SRI-158 and -305). In the blotter paper assay, no charcoal rot infection was observed in SRI-156-treated sorghum roots, indicating complete inhibition of the pathogen, while the roots treated with the other isolates showed 49–76% lesser charcoal rot infection compared to the control. In the antifungal activity test (in green house on sorghum), all the isolates increased shoot dry mass by 15–23% and root dry mass by 15–20% (except SRI-158 and -360), over the control. In order to confirm the plant growth-promoting (PGP) traits of the isolates, the green house experiment was repeated but, in the absence of M. phaseolina. The results further confirmed the PGP traits of the isolates as evidenced by increases in shoot and root dry mass, 22–100% and 5–20%, respectively, over the control. The sequences of 16S rDNA gene of the isolates SRI-156, -158, -178, -211, -229, -305 and -360 were matched with Pseudomonas plecoglossicida, Brevibacterium antiquum, Bacillus altitudinis, Enterobacter ludwigii, E. ludwigii, Acinetobacter tandoii and P. monteilii, respectively in BLAST analysis. This study indicates that the selected bacterial isolates have the potential for PGP and control of charcoal rot disease in sorghum.     

Relation Entre la Teneur en Sucres Réducteurs des Tissus du Tournesol (Helianthus annuus L.) et leur Invasion par Macrophomina phaseolina (Tassi) Goid.

Relation between the amount of reducing sugars in sunflower tissues and their invasion by Macrophomina phaseolina (Tassi) Goid. Reducing sugars (R.S.) were measured with Bernfeld colorimetric method. In the roots, the amount of R.S. increased up to a maximum, then decreased from the beginning of flowering, and remained constant, at a very low level, from the outset of seed maturation. The same evolution was observed, with a delay of one week, in stem bases, but the amounts of R.S. were a little higher. Roots, and thereafter, stems, were invaded by Macrophomina phaseolina only after the minimum level of R.S.was reached. Varietal differences were observed, in a collection of sunflower hybrids, in response to invasion by M. phaseolina. R.S. levels in stem bases were higher in the resistant than in the susceptible hybrids.     

Biology,Epidemiology and Management of the Pathogenic Fungus Macrophomina phaseolina (Tassi) Goid with Special Reference to Charcoal Rot of Soybean (Glycine max (L.) Merrill)

The fungus Macrophomina phaseolina is a causative agent of diseases in more than 500 plant species. The fungus is primarily soil‐inhabiting but is also seed‐borne in many crops including soybean. It survives in the soil mainly as microsclerotia that germinate repeatedly during the crop‐growing season. Low C : N ratio in the soil and high bulk density as well as high soil moisture content adversely affect the survival of sclerotia. The disease can be managed to some extent by cultural practices, organic amendments, seed treatment and genetic host resistance. The scattered literature on these aspects is reviewed in this paper.     

Plant growth-promotion (PGP) activities and molecular characterization of rhizobacterial strains isolated from soybean (Glycine max L. Merril) plants against charcoal rot pathogen, Macrophomina phaseolina

Charcoal rot disease, caused by the fungus Macrophomina phaseolina, leads to significant yield losses of soybean crops. One strategy to control charcoal rot is the use of antagonistic, root-colonizing bacteria. Rhizobacteria A₅F and FPT₇21 and Pseudomonas sp. strain GRP₃ were characterized for their plant growth-promotion activities against the pathogen. Rhizobacterium FPT₇21 exhibited higher antagonistic activity against the pathogen on dual plate assay compared to strain A₅F and GRP₃. FPT₇21 and GRP₃ gave decreased disease intensity in terms of average number of pathogen-infested plants. Lipoxygenase (LOX), phenylalanine ammonia-lyase (PAL), and peroxidase (POD) activities were estimated in extracts of plants grown from seeds that were treated with rhizobacteria, and inoculated with spore suspension of M. phaseolina. The activity of these enzymes after challenge with the test pathogen increased. Strains FPT₇21 and GRP₃ exhibited maximum increases in LOX, PAL and POD activity (U mg⁻¹ fresh leaf wt) compared to strain A₅F.     

Pseudomonas aeruginosa (GRC1) as a strong antagonist of Macrophomina phaseolina and Fusarium oxysporum

A plant growth promotory bacterial strain, isolated from the potato rhizosphere, was characterized as Pseudomonas aeruginosa (GRC1). The isolate produced an hydroxamate type of siderophore after 48 h of incubation on tryptic soy medium under iron deficient conditions. The in vitro antifungal activity of P. aeruginosa was tested against two soil-borne plant pathogens, Macrophomina phaseolina and Fusarium oxysporum. The antagonistic behaviour of the isolate was tested by dual culture technique. The growth inhibition of M. phaseolina and F. oxysporum was 74.1% and 70.5%, respectively, after 5 days of incubation. The production of hydrocyanic acid and indole acetic acid was also recorded under normal growth conditions.     

Peroxidase and Poly-Phenol Oxidase in Brassica juncea Plants Infected with Macrophomina phaseolina (Tassai) Goid. and their Implication in Disease Resistance

Studies on the peroxidase and poly-phenol oxidase activity in relatively resistant and susceptible strains of Brassica juncea at sequential intervals after inoculation with different isolates of Macrophomina phaseolina revealed that, fungus infected tissues had relatively higher activities of these oxidative enzymes in comparison to healthy plants, and tissues inoculated with ‘Weakly Virulent’ isolates always exhibited higher activity in comparison to ‘Moderately’, ‘Highly’ and ‘MostHighly Virulent’ isolates. At the early stages of the infection on 6th day, resistant plants showed higher activities in comparison to their susceptible counterparts. Very low activity of these enzymes could be recorded in healthy plants. Findings of this, study indicate the possible involvement of these oxidative enzymes in the resistance of this disease.     

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:: lacZ 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:: lacZ 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:: lacZ 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.     

Plant growth promotion and suppression of charcoal‐rot fungus (Macrophomina phaseolina) in velvet bean (Mucuna pruriens L.) by root nodule bacteria

Root‐nodulating bacteria are intimate associates of legumes. From a pool of rhizobia isolated from root nodules of Mucuna pruriens (Velvet bean/Kaunch), RMP66 and BMP17 were found to be capable of promoting siderophore and IAA production and phosphate solubilization (insoluble tri‐calcium). Both symbionts were studied further to determine their abilities to promote plant growth and to control root‐rot in Mucuna pruriens caused by the pathogenic plant fungus Macrophomina phaseolina. RMP66 and BMP17 were selected based on their excellent inhibitory activities against M. phaseolina (by 78% and 71%, respectively) in dual culture and in agar‐well assays using cell‐free culture filtrate (CFCF) (by 76% and 62%, respectively). Both strains inhibited fungal growth to a greater extent in iron‐deficient medium (51% and 69%) than in iron‐supplemented medium (37% and 0%), respectively. CFCFs of RMP66 and BMP17 obtained from Pikovskaya's broth and tryptophan‐amended YEM broth inhibited fungal growth by 80%‐55% and 70%‐43%, respectively, and were identified as Sinorhizobium meliloti RMP66 and Bradyrhizobium diazoefficiens BMP17 by 16S rDNA sequencing. Centrifuged and pelleted cells harvested from exponentially grown cultures of S. meliloti RMP66 and B. diazoefficiens BMP17 were used to bacterize seeds of M. pruriens, which then showed enhanced seed germination (by up to 17% and 12%, respectively), and subsequent increases in other plant growth parameters in field trials. Considerable increases in seedling vigour indices (62%: 53% and 110%: 130%) and biomass (8%: 13% and 25%: 28%) were also observed for bacterial treatments. Tn5‐mediated antibiotic‐resistant marker strains showed enhanced nodule occupancy by up to 72% and 68%, respectively. This study describes a multifunctional legume nodule rhizobia that could be utilized in multicropping systems under different agroclimatic conditions as a bioinoculant and alternative to fertilizers.     

Phymatotrichum (cotton) root rot caused by Phymatotrichopsis omnivora: retrospects and prospects

Phymatotrichum (cotton or Texas) root rot is caused by the soil‐borne fungus Phymatotrichopsis omnivora (Duggar) Hennebert. The broad host range of the fungus includes numerous crop plants, such as alfalfa and cotton. Together with an overview of existing knowledge, this review is aimed at discussing the recent molecular and genomic approaches that have been undertaken to better understand the disease development at the molecular level with the ultimate goal of developing resistant germplasm. Taxonomy: Phymatotrichopsis omnivora (Duggar) Hennebert [synonym Phymatotrichum omnivorum (Shear) Duggar] is an asexual fungus with no known sexual stage. Mitosporic botryoblastospores occasionally form on epigeous spore mats in nature, but perform no known function and do not contribute to the disease cycle. The fungus has been affiliated erroneously with the polypore basidiomycete Sistotrema brinkmannii (Bres.) J. Erikss. Recent phylogenetic studies have placed this fungus in the ascomycete order Pezizales. Host range and disease symptoms: The fungus infects most dicotyledonous field crops, causing significant losses to cotton, alfalfa, grape, fruit and nut trees and ornamental shrubs in the south‐western USA, northern Mexico and possibly parts of central Asia. However, this fungus does not cause disease in monocotyledonous plants. Symptoms include an expanding tissue collapse (rot) of infected taproots. In above‐ground tissues, the root rot results in vascular discoloration of the stem and rapid wilting of the leaves without abscission, and eventually the death of the plant. Characteristic mycelial strands of the pathogen are typically present on the root's surface, aiding diagnosis. Pathogenicity: Confocal imaging of P. omnivora interactions with Medicago truncatula roots revealed that infecting hyphae do not form any specialized structures for penetration and mainly colonize cortical cells and eventually form a mycelial mantle covering the root's surfaces. Cell wall‐degrading enzymes have been implicated in penetration and symptom development. Global gene expression profiling of infected M. truncatula revealed roles for jasmonic acid, ethylene and the flavonoid pathway during disease development. Phymatotrichopsis omnivora apparently evades induced host defences and may suppress the host's phytochemical defences at later stages of infection to favour pathogenesis. Disease control: No consistently effective control measures are known. The long‐lived sclerotia and facultative saprotrophism of P. omnivora make crop rotation ineffective. Chemical fumigation methods are not cost‐effective for most crops. Interestingly, no genetic resistance has been reported in any of the susceptible crop species.     

Suppression of root rot of mung bean (Vigna radiata L.) by Streptomyces sp. is associated with induction of peroxidase and polyphenol oxidase

Five strains of Streptomyces sp. were evaluated in vitro for their ability of inhibiting the mycelial growth of Macrophomina phaseolina, the causal agent of root rot of mung bean (Vigna radiata L.). Among the Streptomyces sp. strains tested, PDK showed the maximum in vitro inhibition of mycelial growth of M. phaseolina and recorded an inhibition zone of 21?mm. The strains CBE, MDU, SA and ANR recorded inhibition zones of 18, 16, 13 and 11?mm, respectively. These Streptomyces sp. strains were tested for their growth-promoting efficiency on mung bean seedlings. Among them, CBE and PDK recorded the maximum increase in shoot length, root length and seedling vigour compared with control, followed by MDU. Three Streptomyces sp. strains (CBE, MDU and PDK) that showed higher levels of inhibition of growth of M. phaseolina in dual culture assay and plant growth-promoting activity were tested for their biocontrol activity against root rot under greenhouse and field conditions. Seed treatment or soil application with powder formulation of Streptomyces sp. strains CBE, MDU and PDK was effective in controlling root rot disease; but, combined application through seed and soil increased the efficacy in both the greenhouse and field trials. Among the treatments, seed treatment plus soil application with powder formulation of Streptomyces sp. strain CBE proved to be most effective, which reduced the root rot incidence from 26.8% (with non-bacterised seeds) to 4.0% in Trial I and from 32.0 to 4.9% in Trial II. The above treatment recorded the highest yield in both the field trials, and the yield increase was 78 and 74% over control in Trial I and Trial II, respectively. Isozyme analysis of the Streptomyces sp.-treated plants indicates that seed treatment plus soil application strongly induce the activities of peroxidase (PO-1 and PO-2) and polyphenol oxidase (PPO-2 and PPO-3) in mung bean. Among the three strains tested, Streptomyces sp. strain MDU- treated plants showed higher levels of activities of PO and PPO. Based on the above findings, it can be concluded that both the direct inhibition of pathogen and induced resistance might be involved in the control of root rot of mung bean by Streptomyces sp.     

Quantitative trait locus responsible for resistance to Aphanomyces root rot (black root) caused by Aphanomyces cochlioides Drechs. in sugar beet

Aphanomyces root rot, caused by Aphanomyces cochlioides Drechs., is one of the most serious diseases of sugar beet (Beta vulgaris L.). Identification and characterization of resistance genes is a major task in sugar beet breeding. To ensure the effectiveness of marker-assisted screening for Aphanomyces root rot resistance, genetic analysis of mature plants’ phenotypic and molecular markers’ segregation was carried out. At a highly infested field site, some 187 F₂ and 66 F₃ individuals, derived from a cross between lines ‘NK-310mm-O’ (highly resistant) and ‘NK-184mm-O’ (susceptible), were tested, over two seasons, for their level of resistance to Aphanomyces root rot. This resistance was classified into six categories according to the extent and intensity of whole plant symptoms. Simultaneously, two selected RAPD and 159 ‘NK-310mm-O’-coupled AFLP were used in the construction of a linkage map of 695.7 cM. Each of nine resultant linkage groups was successfully anchored to one of nine sugar beet chromosomes by incorporating 16 STS markers. Combining data for phenotype and molecular marker segregation, a single QTL was identified on chromosome III. This QTL explained 20% of the variance in F₂ population (in the year 2002) and 65% in F₃ lines (2003), indicating that this QTL plays a major role in the Aphanomyces root rot resistance. This is the first report of the genetic mapping of resistance to Aphanomyces-caused diseases in sugar beet.     

Candida pruni sp. nov. is a new yeast species with antagonistic potential against brown rot of peaches

Brown rot caused by Monilinia spp. is among the most important postharvest diseases of commercially grown stone fruits, and application of antagonistic yeasts to control brown rot is one promising strategy alternative to chemical fungicides. In this research, new yeast strains were isolated and tested for their activity against peach brown rot caused by Monilinia fructicola. Three yeast strains were originally isolated from the surface of plums (cv Chinese Angelino) collected in the north of China. In artificially wounded inoculation tests, the yeast reduced the brown rot incidence to 20 %. The population of the yeast within inoculated wounds on peaches significantly increased at 25 °C from an initial level of 5.0 × 10⁶ to 4.45 × 10⁷ CFU per wound after 1 day. The antagonistic strains were belonging to a new species of the genus Candida by sequence comparisons of 26 S rDNA D1/D2 domain and internal transcribed spacer region. The strains are most closely related to C. asparagi, C. musae and C. fructus on the basis of the phylogenetic trees based on the D1/D2 region of 26S rDNA. However, the strains are notably different from C. asparagi, C. musae and C. fructus, in morphological and physiological characteristics. Therefore, the name Candida pruni is proposed for the novel species, with sp-Quan (=CBS12814^T = KCTC 27526^T = GCMC 6582^T) as the type strain. Our study showed that Candida pruni is a novel yeast species with potential biocontrol against brown rot caused by M. fructicola on peaches.