Screening of bacteria of the genus Bacillus for the control of the plant-pathogenic fungus Macrophomina phaseolina

This study evaluated the efficiency of 19 Bacillus isolates, obtained from the rhizosphere and rhizoplane of wild and cultivated castor bean plants, to control the pathogenic fungus Macrophomina phaseolina. Using in vitro assays, we examined the antifungal effects of volatile and non-volatile compounds, the production of siderophores and the activity of chitinase in these isolates. In vivo experiments were conducted to determine the potential of the Bacillus isolates to colonise castor bean plant roots and to control the fungus. In general, results showed that isolates from wild castor bean, compared with isolates from cultivated castor bean, were more efficient producers of antifungal compounds, better colonisers of plant roots and more effective protectors of plant seedlings against infection with M. phaseolina. Altogether, isolate RP 5, originating from the rhizoplane of wild castor bean, was the most promising candidate for future evaluation as a biological control agent of M. phaseolina.     

Molecular Genotyping of Macrophomina phaseolina Isolates: Comparison of Microsatellite Primed PCR and Repetitive Element Sequence-based PCR

Seventy isolates of Macrophomina phaseolina recovered from different host plants were assessed for DNA polymorphism using two molecular techniques: microsatellite primed polymerase chain reaction (MSP‐PCR) under both touchdown (T) and non‐touchdown (NT) PCR conditions and primers corresponding to disperse repetitive sequence‐based polymerase chain reaction (rep‐PCR). Fingerprints obtained by rep‐PCR were compared with those of MSP‐PCR. Even though these methods yielded intraspecific polymorphisms, yet different levels of discrimination could be obtained. A partial correlation was apparent between the molecular techniques used. Some of the genetic groups/genotypes were supported by both the molecular markers employed in the study, thus confirming their relationship. Thirty nine MSP (T), 55 MSP (NT) and 53 rep‐PCR genotypes were identified with discrimination indices of 0.962, 0.993 and 0.99, respectively. Our results have shown that rep‐PCR is a rapid, inexpensive technique that is highly reproducible and almost as discriminatory as MSP‐PCR for genotyping M. phaseolina isolates and is highly suitable for understanding disease epidemiology at molecular level. Suggesting, thereby, that it is a robust technique employed for genotypical and phylogenetic studies for determining taxonomical diversity and phylogenetic structure of the economically important fungal pathogen of cluster bean. The data presented here will help researchers to design effective strategies for deployment of resistant germplasm in cluster bean (Cymopsis tetragonoloba) growing regions in the country and worldwide.     

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.     

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.     

Effect of Irrigation Type on Inoculum Density of Macrophomina phaseolina in Melon Fields in Arizona

Charcoal rot, caused by Macrophomina phaseolina, has become increasingly problematic for melon growers using subsurface drip irrigation in Arizona; but has rarely been observed in fields with furrow irrigation. Since the relationship between increasing incidence of charcoal rot on melon and irrigation type is unknown, studies were initiated to determine the effects of edaphic factors on inoculum density. Soil samples were collected once from fields irrigated by subsurface drip, with and without plastic mulch, and by furrow at 10, 20 and 30 cm depths. Samples were analysed for percentage soil moisture, pH, salinity and inoculum density. Percentage soil moisture was significantly higher at 20 and 30 cm depths in the furrow‐irrigated field compared with the drip‐irrigated field with plastic mulch, but not in the field without plastic mulch. Average minimum and maximum temperatures and inoculum density were significantly lower at all three depths in the furrow‐irrigated field compared with both types of drip irrigation. pH was significantly higher in the furrow‐irrigated field compared with both types of drip irrigation at 20 and 30 cm depths but not at 10 cm depth. Differences in inoculum densities of M. phaseolina suggest that drip irrigation may contribute to higher disease incidences.     

Occurrence of Charcoal Rot Caused by Macrophomina phaseolina,an Emerging Disease of Adzuki Bean in China

Adzuki bean (Vigna angularis) is an important legume crop in China. Soil‐borne charcoal rot caused by Macrophomina phaseolina (Tassi) Goid is an important and devastating disease of many crops including legumes. During late August and early September, 2014, symptoms similar to charcoal rot were observed on adzuki bean plants in Yulin City of Shanxi Province, and Fangshan County of Beijing, China. This study was conducted to determine the causal agent of the emerging disease on adzuki bean. Four fungal isolates were obtained and identified as M. phaseolina based on morphological and molecular characteristics, including species‐specific primer detection and sequences of internal transcribed spacer (ITS) of nuclear ribosomal DNA. The resulting sequences showed 99% identity with more than 60 M. phaseolina strains from diverse hosts. The virulence on adzuki bean was verified using pathogenicity tests, producing symptoms similar to those observed in the fields. To our knowledge, this is the first report of M. phaseolina causing charcoal rot on adzuki bean.     

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.     

The influence of mineral and carbon sources on biological control of charcoal rot fungus,Macrophomina phaseolina by fluorescent pseudomonads in tomato

AIMS: To determine the influence of various trace minerals and carbon source on the biocontrol performance of Pseudomonas aeruginosa strain IE-6S+ and P. fluorescens strain CHA0 against Macrophomina phaseolina. METHODS AND RESULTS: In dual culture plate assay, P. aeruginosa IE-6S+ and P. fluorescens CHA0 inhibited radial growth of M. phaseolina producing zones of inhibition. Czapek's dox agar medium amended with both zinc and glucose remarkably improved antifungal activities of the bacterial inoculants. Under glasshouse conditions, soil amendment with zinc and/or glucose alone did not reduce M. phaseolina infection in tomato roots but did reduce significantly when used in combination with IE-6S+ or CHA0. Soil amendments with zinc and/or glucose increased fresh shoot weights but zinc amendment greatly reduced bacterial populations in the rhizosphere. CONCLUSIONS: Mineral and carbon amendments enhance the biocontrol performance of fluorescent pseudomonads against M. phaseolina. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of mineral and carbon amendments that favour biocontrol of certain bacterial strains may provide clues to soil factors or components of nutrient solutions in hydroponic culture that will improve the level and reliability of control.     

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.     

Macrophomina phaseolina alters the biochemical pathway in Vigna radiata chastened by Zn2+ and FYM to improve plant growth

Mung bean [Vigna radiata (L.) Wilczek] is an important cash pulse crop extensively cultivated in the arid region of Pakistan, which encounters intimidating charcoal rot disease caused by Macrophomina phaseolina (Tassi) Goid. The current research was conducted to check the potential of Zn (1.25, 2.44 and 5?mg?kg^?1) and FYM [farmyard manure (1% and 2%)] in mono-, bi- and trilateral interaction in managing disease and improving yield. Suppression of plant immunity by M. phaseolina was indicated by the change in activities of antioxidant enzymes (CAT and SOD) and cell wall strengthening enzymes (POX and PAL) that revealed inability of the protein receptor to identify the pathogen elicitor. FYM improved soil physicochemical properties and beneficial microbes activity, which released antimicrobial protein- and plant defense-stimulating protein and in response to ROS (reactive oxygen species) signaling molecules plant susceptibility was reduced. However, Zn as a co-factor chastened the ROS in stressed cells by upregulation of antioxidant enzymes in favor of the plant. The complex interaction of FYM and Zn potentially hijacked the further multiplication of pathogen. Finally, soil amendment improved biological attributes and grain yield to profitable farming in terms of harvest index percentage and benefit–cost ratio.     

The effect of high temperature and fallow period on infection of mung bean and cashew roots by the vesicular-arbuscular mycorrhizal fungus Glomus intraradices

The experiments described were designed to investigate the way in which high temperatures (30°C and above) affected the survival and infectivity of spores of Glomus intraradices formulated as the commercial inoculum NutriLink^TM. Infection of mung bean (Vigna radiata) occurred most rapidly at 30°C compared with either 22° or 38°C, although the final percentage of the root length infected (6 weeks) was similar at all three temperatures. Early rapid infection led to greater plant growth of this species at 30°. In cashew (Anacardium occidentale) no infection occurred at 38°C and this was associated with low plant growth, compared with the other temperatures at which infection reached 40–60% after 4 months. In both species differences in root temperature were associated with marked differences in the morphology and growth of the root systems, with poor root growth at 38°C. Spores of G. intraradices retained infectivity with respect to mung bean for up to 6 weeks in moist fallow soil, although maximum infectivity was observed in soil without a fallow period. The effects of temperature on germination of spores buried in filter paper sandwiches in soil were consistent with the data for infection and growth. Germination was most rapid and reached the highest percentage at 3 weeks at 30°C. Lowest germination was attained at 38°C. We conclude that G. intraradices can retain its infectivity in moist soil at high temperatures, but that the extent to which the plants become infected and hence their response, depends not only on this but also on host factors such as root growth.     

Synergistic effects of plant growth retardants and IBA on the formation of adventitious roots in hypocotyl cuttings of mung bean

The synergistic effect of plant growth retardants, such as daminozide, paclobutrazol and triadimefon, and of indole-3-butyric acid (IBA) on the formation of adventitious roots in hypocotyl cuttings of mung bean was studied. The three retardants and IBA all stimulated adventitious root growth, but IBA was the most effective. However, mixtures of the retardants with IBA have proven generally more effective than IBA alone in promoting adventitious root formation. When IBA was applied to the hypocotyls one day after cutting preparation followed by the growth retardant on the second day, there were even more adventitious roots produced than if applied in the reverse order. The effectiveness of the treatments were in the order, IBA followed by growth retardant, IBA + growth retardant together, and IBA alone.Abbreviations IBA indole-3-butyric acid - GA gibberellin     

Studies on the relationship between cyanide-resistant respiration and expression of alternative oxidase in mung bean using antibodies prepared by synthetic polypeptide

Twelve peptides, including eight conservative amino acid residues in the amino acid sequence of hydrophilic S helix of the alternative oxidase (AOX), were synthesized by solid-phase method. The polypeptide was coupled with α-chymotrypsinogen, and the antibodies were obtained through immunizing domestic rabbit by injecting this complex. By using these antibodies, which were raised to immunoreact with total proteins of purified mitochondria from different organs of mung bean seedlings, we find that there are two hybridizable AOX bands in mitochondria. Their molecular weights are about 35 and 38 ku, respectively. Moreover, the respiratory parameters of hypocotyl, true leaf and cotyledon of mung bean seedlings show that true leaf has the highest total respiration (V_t), alternative pathway (AP) capacity (V_alt) and the activity of AP (ρV_alt) among the three organs. V_t andρV _alt of cotyledon ranked the second. Hypocotyl has the lowest V_t andρV _alt, but its V_alt is higher than that of cotyledon. These results are consistent with the analysis of Western blotting of expression of AOX. The highest V_t andρV _alt in true leaf are accompanied two hybridizable polypeptides of AOX protein, 35 ku and 38 ku respectively. The next is cotyledon V_t andρV _alt with only one 38 ku hybridizable polypeptide of AOX protein. HypocotylV _t andρV _alt is the lowest and its immunoblotting band is similar to that of cotyledon, but the expressive amount of 38 ku protein is less than that of cotyledon. The results suggest that the 35 ku AOX may contribute mainly to true leafρV _alt.     

Studies on the relationship between cyanide-resistant respiration and expression of alternative oxidase in mung bean using antibodies prepared by synthetic polypeptide

Twelve peptides, including eight conservative amino acid residues in the amino acid sequence of hydrophilic S helix of the alternative oxidase (AOX), were synthesized by solid-phase method. The polypeptide was coupled with α-chymotrypsinogen, and the antibodies were obtained through immunizing domestic rabbit by injecting this complex. By using these antibodies, which were raised to immunoreact with total proteins of purified mitochondria from different organs of mung bean seedlings, we find that there are two hybridizable AOX bands in mitochondria. Their molecular weights are about 35 and 38 ku, respectively. Moreover, the respiratory parameters of hypocotyl, true leaf and cotyledon of mung bean seedlings show that true leaf has the highest total respiration (Vt), alternative pathway (AP) capacity (Valt) and the activity of AP (ρValt) among the three organs. Vt and ρValt of cotyledon ranked the second. Hypocotyl has the lowest V, and ρValt, but its Valt is higher than that of cotyledon. These result