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.     

Hydroxylation of (+)-menthol by Macrophomina phaseolina

Abstract

Biotransformation of (+)-menthol with Macrophomina phaseolina led to hydroxylations at C-1, C-2, C-6, C-7, C-8 and C-9, with the C-8 position being preferentially oxidized. The resulting metabolites were identified as 8-hydroxymenthol (2), 6R-hydroxymenthol (3), 1R-hydroxymenthol (4), 9-hydroxymenthol (5), 2R,8-dihydroxymenthol (6), 8S,9-dihydroxymenthol (7), 6R,8-dihydroxymenthol (8), 1R,8-dihydroxymenthol (9) and 7,8-dihydroxymenthol (10). Metabolites 6–10 are described here for the first time. Their structures were characterized by spectroscopic analysis.     

Intraspecies diversity of Macrophomina phaseolina in Iran

To study the pathogenic and genetic diversity of the Macrophomina phaseolina in Iran, 52 isolates of the fungus were isolated from 24 host plants across the 14 Iranian provinces. All isolates were confirmed to the species based on the species-specific primers. The aggressiveness of M. phaseolina isolates was evaluated on the common bean. Based on the pathogenicity tests, M. phaseolina isolates from the different hosts displayed different levels of aggressiveness on the common beans. The results showed that there was significant variation in the aggressiveness of the pathogen; however, there was no distinct pattern of differentiation based on the host or geographical origin linked to the virulence of the isolates, as frequently theisolates from the same host or geographical origin had different levels of aggressiveness. Inter-simple sequence repeat (ISSR) markers were used to assess the genetic diversity of the fungus. The unweighted pair-group method, using arithmetic mean clustering of data, showed that isolates did not clearly differentiate to the specific group according to the host or geographical origins; however, usually the isolates from the same host or the same geographical origin tend to group nearly. Our results did not show a correlation between the genetic diversity based on the ISSR and pathogenic patterns on common bean in the greenhouse. Similar to the M. phaseolina populations in the other countries, the Iranian isolates were highly diverse based on the pathogenic and genotypic characteristics.     

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.     

A New Stem-splitting Symptom in Safflower Caused by Macrophomina phaseolina

Safflower is known to be attacked by several seed‐borne fungi, of which Macrophomina phaseolina is one of the most important pathogens causing serious yield losses. During routine experiments, a new stem‐split symptom was observed in M. phaseolina‐infected plants resulting in poor growth and reduced size of inflorescences. Stem‐split was observed in 30‐day‐old plants as minute cracks approximately 2–3 cm above the soil surface, which over time extended to both upward and downward directions, resulting in the formation of a wider split. The split portion was hollow and brown with a white to grey mycelial mat of the fungus on the inner surface. Such plants became lodged, ultimately resulting in poor seed yield compared with healthy plants. The stem‐split plants showed delayed flowering by 1 week over healthy plants. One of the three M. phaseolina isolates used for inoculation of seeds and plants was more aggressive but all isolates were able to reproduce the stem‐split symptoms found on naturally infected safflower plants in the field.     

Effect of root-rot fungus (Macrophomina phaseolina) on the life cycle of root-knot nematode (Meloidogyne javanica) Race-2 on balsam

The penetration of second stage juveniles of Meloidogyne javanica started within 12 hours after inoculation and the rate of penetration gradually increased with the passage of time up to the fifth day in the plants inoculated with root-knot nematode alone and up to the sixth day when plants were infected with root-knot nematode and root-rot fungus. Mostly, the penetration of second stage juveniles of Meloidogyne javanica took place in the meristematic region but in some cases the juveniles also penetrated into the root tips and oriented themselves near the stellar region almost parallel to the longitudinal axis of the roots. The life cycle of Meloidogyne javanica on balsam was completed within 25 days, whereas the duration of the life cycle and fecundity of females was adversely affected in the presence of fungus (Macrophomina phaseolina) and it took about 33 days to complete the life cycle, i.e. the presence of Macrophomina phaseolina delayed the life cycle of the root-knot nematode (Meloidogyne javanica) by eight days.     

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.     

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.     

Improvement of antagonistic capability of Trichoderma harzianum by UV-Irradiation for management of Macrophomina phaseolina

Antagonistic capability of Trichoderma harzianum was improved through UV-irradiation. Four different type of mutants, T. harzianum - M_a (Th-M_a), T. harzianum - M_b(Th-M_b), T. harzianum - M_c (Th-M_c), T. harzianum - M_d (Th-M_d) of T. harzianum and the parent strain (Th-P) were selected for further studies. Th-M_a and Th-M_b showed more antagonistic capability against Macrophomina phaseolina than its parent strain Th-P in dual culture. Biochemical analysis of these four mutants and the parent strain showed that Th-M_a releases higher level of two lytic enzymes i.e. chitinases and cellulases and Th-M_b produces more β-1,3-glucanase activity than the parent strain. Culture filtrate of Th-M_a also showed antifungal properties. Study of the competitive saprophytic ability (CSA) of these four mutants and the parent strain were also made. Th-M_a exhibited higher CSA than the parental isolate while Th-M_d had less CSA than all other mutants and the parent strain of T. harzianum.     

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.     

Development of a Sensitive Serological Method for Specific Detection of Latent Infection of Macrophomina phaseolina in Cowpea

A double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific detection and quantification of Macrophomina phaseolina in plant tissue. Both polyclonal antisera produced against immunogens from mycelium and culture filtrate of M. phaseolina detected the fungus in mycelial and plant extracts, although the antibodies raised against mycelium were more sensitive. No cross-reaction occurred with Rhizopus stolonifer , Pythium ultimum , Mucor hiemalis , Fusarium oxysporum , Septoria nodorum , Rhizoctonia solani , Sclerotinia sclerotiorum , Phytophthora infestans and Aspergillus niger . In enzyme assays, activity of the endo-acting hydrolytic enzymes 1,3-β-glucanase and, less, cellulase, but not xylanase was detected in infected plants. DAS-ELISA was more sensitive than the 1,3-β-glucanase assay. In polyacrylamide gel electrophoresis (PAGE) up to 18 protein bands were observed, with four bands occurring in the 12 tested isolates deriving from various geographical origin in Niger and Nigeria. The enzyme assays and protein patterns were considered not suitable for specific M. phaseolina detection. Macrophomina phaseolina was essentially located in the roots and hypocotyls, and less in epicotyls and leaves of infected plants. The antibodies were also useful to detect latent infection and the infection of cowpea seeds.     

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.     

Detection,identification and morphological characteristic of Macrophomina phaseolina: the charcoal rot disease pathogens isolated from infected plants in Northern Jordan

This is the first report about charcoal rot disease in Jordan. Twenty-five Macrophomina phaseolina isolates were collected from infected plants showing typical symptoms of charcoal rot disease. All of the 25 M. phaseolina isolates were pathogenic to cucumber plants under green house effect. The amplification of the isolated DNA from the 25 pathogenic fungal cultures using ITS specific primers (ITS 1?+?ITS 4) showed a single band of 580?bp. There was a significant variation of their mycelial linear growth rate on PDA medium. The 25 M. phaseolina isolates showed a wide heterogeneity in their mycelium colour, microsclerotia distribution, pycnidia formation and chlorate phenotypes. Based on the morphological characterisation, the 25 isolates were grouped into seven different groups as indicated in a dendrogram of their morphological variation. The overall means similarity matrix of the 25 M. phaseolina recovered isolates were 0.58. The means of similarity matrix of the 25 M. phaseolina was in between 0.83 and 0.14. The similarity coefficient between the 25 isolates varies between 0.27 and 1.0.     

Temperature response,pathogenicity, seed infection and mutant evaluation against Macrophomina phaseolina causing charcoal rot disease of sesame

Charcoal rot caused by Macrophomina phaseolina is a serious disease of sesame in Pakistan. M. phaseolina sesame isolate was subjected to growth rate test at 10, 15, 20, 25, 30, 35 and 40°C. The optimum temperature for fungal growth and microsclerotia production was found to be 30–35°C. Gray to black, radial fungal colonies with intermediate mycelial growth and jet black oval to round microsclerotia were observed at this optimum range. M. phaseolina was found to be pathogenic against all the 18 tested plant species and this pathogenicity proved its necrophytic behavior. Seed infection efficiency of M. phaseolina was 100% with significant reduction in seed index. For two consecutive years 21 mutants/varieties were screened in the field for their reactions to charcoal rot disease. During 2007 three mutants NS11704S1, NS11304S2 and NS26004 were ranked as resistant while others were moderately resistant to highly susceptible. During 2008 all mutants showed a susceptible to highly susceptible reaction with variable disease reactions. All over screening results revealed that four mutants viz, NS13P1, NS163-1, NS270P1 and NS26004 showed about 50% stand with consistent performance during both years under optimum disease conditions and can be used to manage the disease following the disease management strategies, however in the future improvement for high seed yield along with resistance is a prerequisite for sustainable high production.     

Morphological and pathogenic variability in Macrophomina phaseolina isolates of pigeonpea (Cajanus cajan L.) and their relatedness using principle component analysis

Sixty-one isolates of Macrophomina phaseolina were characterised on the basis of different morphological characters to study the variability in the population. PCA analysis extracted three main components, microsclerotia, texture and colour, from the population that described the variability in the population most appropriately. Colour of the isolates and the presence of the microsclerotia have a significant effect on the area under disease progress curve. Isolates with the production of microsclerotia (M⁺) were more aggressive as compared to isolates with no production of microsclerotia (M^?). The study has brought out pathogenic variation in M. phaseolina, thus better understanding of host pathogen relationship to identify physiologic races in the breeding programme.     

Development of linkage map in F2 population of selected parents with respect to Macrophomina phaseolina resistance trait using screened polymorphic RAPD and developed SCAR markers of jute

Two molecular markers (RAPD and simple sequence repeat (SSR)) were applied on 12 Corchorus capsularis jute samples. Two of them were Macrophomina phaseolina-resistant and the remaining eight were M. phaseolina-susceptible accessions. Eleven SSR primer combinations out of 18 gave the polymorphic results between M. phaseolina-resistant and -susceptible accessions. Five pairs of sequence characterised amplified region (SCAR) primers designated as SCP-4, SCS-3, SCS-13, SCG-10 and SCU-10 were designed based on the polymorphic loci obtained between JRC-412 and CIM-036. Only SCU-10 and SCS-13 produced polymorphic markers corresponding to OPU-10 and OPS-13 amplified from ‘CIM-036’ and JRC-412, respectively. RAPD and SCAR markers were employed for construction of a linkage map using a set of 67 F2 population of a cross between JRC-412 and CIM-036 as a mapping population. Nine markers were assigned to two linkage groups (LGs) covering a total length of 628.4 cM with an average distance of 28 cM between markers.     

Management of disease complex of balsam caused by Meloidogyne javanica and Macrophomina phaseolina by using biofertilisers and pesticides

An investigation was conducted to study the effect of biofertilisers (Glomus fasciculatum, Azospirillum brasilense, Azotobacter chroococcum and Micro Phos) and pesticides (carbofuran and bavistin) on the management of root-rot and root-knot disease complex of balsam. The individual application of biofertilisers (G. fasciculatum, A. brasilense and A. chroococcum) significantly improved the plant growth parameters viz., length, dry weight and number of flowers compared to untreated-uninoculated plants. The simultaneous inoculation of plant with Meloidogyne javanica and Macrophomina phaseolina in the pots treated with either of the biofertilisers (G. fasciculatum, A. brasilense and A. chroococcum) and pesticides (carbofuran/bavistin) significantly improved the plant growth parameters and reduced the reproduction factor, number of galls and intensity of root-rot compared to untreated-inoculated plants. On the other hand, the plants treated with Micro Phos neither significantly improved the plant growth parameters nor reduced the reproduction factor, number of galls and intensity of root-rot. Among the biofertilisers and pesticides, carbofuran was found to be the most effective in the management of disease complex of balsam followed by bavistin, G. fasciculatum, A. brasilense and A. chroococcum.