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.     

Genetics of nonsenescence and charcoal rot resistance in sorghum

Summary Nonsenescence is a delayed leaf and plant death resistance mechanism in sorghum that circumvents the detrimental effects of reduced soil moisture combined with high temperatures during post-anthesis growth. This drought-tolerance mechanism is often equated with charcoal rot resistance, a widespread root and stalk disease of great destructive potential. Therefore, the inheritance of charcoal rot resistance was investigated directly, by exposure of sorghum to Macrophomina phaseolina, the causal organism, and indirectly, by determination of the inheritance of nonsenescence. Sorghum families derived from diallel crosses between two nonsenescent, resistant inbreds (B35, SC599-11E) and two senescent, susceptible inbreds (BTx378, BTx623) were evaluated in 1989 at College Station and at Lubbock, Texas, under controlled and field conditions. We determined that nonsenescence was regulated by dominant and recessive epistatic interactions between two nonsenescence-inducing loci and a third locus with modifying effects. The same conclusion was reached for charcoal rot resistance. The presence of different genetic mechanisms within SC599-11E for nonsenescence and charcoal rot resistance verifies that these two forms of resistance are not different manifestations of a single trait, i.e., they are not to be equated with each other. We conclude that nonsenescence alone cannot account for, and should not be used as the sole breeding criterion for, resistance to charcoal rot in sorghum.     

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.     

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.     

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.     

Germination and production ofMacrophomina phaseolina microsclerotia as affected by oxygen and carbon dioxide concentration

Summary Germination of microsclerotia ofMacrophomina phaseolina was observed at O₂ concentrations of 16% or higher in autoclaved soil. Germination was delayed but otherwise unaffected as O₂ decreased from 21 to 16% and was in all cases complete in 32 hours. Laboratory-produced microsclerotia consistently germinated more rapidly and seemed more independent of O₂ concentrations within the range that permitted germination than naturallyproduced microsclerotia.Population changes in soil as measured by microsclerotial counts were inversely correlated with depth of interment and reduced O₂ concentration. Our inability to detect significantly growth responses ofM. phaseolina in non autoclaved soil was apparently related to limited O₂ although other possibilities are discussed.Contribution of the Missouri Agricultural Experiment Station Scientific Journal Series No. 9124.     

Population dynamics of Macrophomina phaseolina in relation to disease management: A review

Macrophomina phaseolina (Tassi) Goid. causes seedling blight, charcoal rot, leaf blight, stem and pod rot on over 500 plant species in different parts of the world. The pathogen survives as sclerotia formed in host tissues which are released into the soil as tissue decay. Low soil moisture is considered the more important predisposing factor for M. phaseolina-induced diseases than high temperature. The intensity of the disease on a crop is related to the population of viable sclerotia in the soil and abiotic factors. The influence of various management strategies in reducing the number of viable propagules of the pathogen in the soil has been studied in order to minimize the impact of the disease. Any management approach that reduces inoculum density in the soil may reduce disease incidence on the host. However, to reduce inoculum density, quantitative determination of viable propagules from soil is necessary in order to understand the effect of management strategies on the population dynamics of this pathogen. Considerable work has been done on organic amendments, changing crop sequences with tolerant crops, fumigants, herbicides and tillage in managing M. phaseolina populations in the soil and the resulting disease. Solarization has been used in controlling M. phaseolina in different countries where this pathogen is causing disease on economically valuable crops. However, this method of soil disinfestation was effective in eliminating viable populations at the top soil layer although by combining other approaches its effectiveness was improved at lower soil depth. Use of biological control agents with or without organic amendments or after solarization has emerged to be a practical management approach in the control of M. phaseolina. In this paper, an attempt has been made to review those research findings where the influence of various management approaches on survival of M. phaseolina mainly sclerotia have been investigated.     

Identification of charcoal rot resistance QTLs in sorghum using association and <Emphasis Type="Italic">in silico</Emphasis> analyses

Charcoal rot disease, a root and stem disease caused by the soil-borne fungus Macrophomina phaseolina (Tassi) Goid., is a major biotic stress that limits sorghum productivity worldwide. Charcoal rot resistance-related parameters, e.g., pre-emergence damping-off%, post-emergence damping-off%, charcoal rot disease severity, and plant survival rates, were measured in a structured sorghum population consisting of 107 landraces. Analysis of variance of charcoal rot resistance-related parameters revealed significant variations in the response to M. phaseolina infection within evaluated accessions. Continuous phenotypic variations for resistance-related parameters were observed indicating a quantitative inheritance of resistance. The population was genotyped using 181 simple sequence repeat (SSR) markers. Association analysis identified 13 markers significantly associated with quantitative trait genes (QTLs) conferring resistance to charcoal rot disease with an R² value ranging between 9.47 to 18.87%, nine of which are environment-specific loci. Several QTL-linked markers are significantly associated with more than one resistance-related parameter, suggesting that those QTLs might contain genes involved in the plant defense response. In silico analysis of four novel major QTLs identified 11 putative gene homologs that could be considered as candidate genes for resistance against charcoal rot disease. Cluster analysis using the genotypic data of 181 SSR markers from 107 sorghum accessions identified 12 main clusters. The results provide a basis for further functional characterization of charcoal rot disease resistance or defense genes in sorghum and for further dissection of their molecular mechanisms.     

Screening of sorghum genotypes and biochemical changes for resistance to damage caused by Macrophomina phaseolina

Charcoal rot, caused by Macrophomina phaseolina, is the most common yield reducing constraint in sorghum. In the present study, eight cultivars of Sorghum bicolor were screened against M. phaseolina to determine its effects on several growth parameters as well as the content of phenols, salicylic acid (SA), total protein and peroxidase activity and were examined for their relationship, with disease resistance. Out of the eight cultivars tested, PJ-1430 was the most resistant and SU-1080, the most susceptible. The roots of plants were more affected by the pathogen than the shoots. The cultivar PJ-1430 produced a higher plant root and shoot dry mass and was associated with higher polyphenols, SA, peroxidases and thus sustained less oxidative damage whereas SU-1080 experienced maximum oxidative damage, so was considered susceptible to charcoal rot. A positive relationship was observed among various biochemical parameters and disease resistance of cultivars PJ-1430 and SU-1080.     

Defense gene expression in Sorghum bicolor against Macrophomina phaseolina in leaves and roots of susceptible and resistant cultivars

The fungal disease, charcoal root rot, caused by Macrophomina phaseolina is a foremost yield restraining factor of Sorghum bicolor L. around the world. The expression analysis of genes induced in general defense response can endow with clues to reveal major defense mechanisms against pathogen infection in sorghum plant. The role of chitinase and Stilbene synthase in response to M. phaseolina in sorghum was studied under control growth conditions using a real-time polymerase chain reaction. Here, we report the expression analysis of antifungal genes in two cultivars viz. PJ-1430 (resistant) and SU-1080 (susceptible) at different hours after inoculation with M. phaseolina isolate MTCC 2165. Chitinase and stilbene synthase were induced in PJ-1430 within 0 h, 24 h and in SU-1080 in 48 h, 24 h, respectively, after inoculation. However, the expression levels of chitinase and stilbene synthase in resistant cultivar were significantly higher. The results showed that chitinase and stilbene synthase can be effective to enhance resistance to M. phaseolina.     

Effect of organic soil amendments on the incidence of stalk rot of maize

Five organic materials, Calopogonium sp. leaves, rice straw, mixed wood saw dust of Terminalia sp. Triplochyton sp. and Kaya sp., fresh guinea grass and poultry manure were added to soil with a recent history of high stalk rot of maize caused by Macrophomina phaseolina and Fusarium moniliforme. The two stalk rot organisms reacted differently to the organic amendments. All the amendments produced significantly less Fusarium stalk rot disease than the unamended control. Amendment of soil with fresh Calopogonium leaves was almost ineffective in controlling the incidence of Macrophomina phaseolina. Organic amendment with poultry manure increased the disease casued by M. phaseolina. The other amendments appeared to be equally effective in checking the disease. Increased microbial population due to amendment may play some role in disease suppression.     

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.     

Potential bio-control agent for management of post-flowering stalk rot complex and maize plant health

The of aim of this study was to evaluate the potentiality of the native isolate of Trichoderma harzianum from IARI farm for the management of post-flowering stalk rot of maize and improvement of plant health. The treatment was tried as a seed treatment with the potent commercial formulation Kalisena SD (Aspergillus niger), neem kernel powder and effective fungicides (Thiram, Carbendazim, Captan) for comparison of its efficacy. The minimum disease was recorded in plots treated with native isolate both due to Fusarium moniliforme and Macrophomina phaseolina. The fungicides Carbendazim and Thiram ranked second in controlling the disease due to F. moniliforme and M. phaseolina, respectively. Plant health with respect to vigour and plant stand were found to be better than those of all the other treatments. It is concluded that the native isolate of T. harzianum may be used for maintaining good plant health and managing post-flowering stalk rot of maize.     

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.     

Carbon and nitrogen dynamics across a natural precipitation gradient in Patagonia,Argentina

Abstract. Both ecosystem carbon gain and nutrient availability are largely constrained by the magnitude and seasonality of precipitation in arid and semi‐arid ecosystems. We investigated the role of precipitation on ecosystem processes along an International Geosphere Biosphere Programme (IGBP) transect in temperate South America. The transect consists of a contiguous precipitation gradient in the southern region of Argentinean Patagonia (44–45° S), from 100 mm to 800 mm mean annual precipitation (MAP) and vegetation ranging from desert scrub to closed canopy forest. Gravimetric soil water content tracked changes in seasonal and annual precipitation, with a linear increase in soil water content with increasing MAP. Above‐ground net primary production (ANPP) increased linearly along the gradient of precipitation (ANPP =– 31.2 + 0.52 MAP, r²= 0.84, p= 0.028), supporting the relationship that carbon assimilation is largely controlled by available water in these sites, and was in general agreement with regional models of ANPP and rainfall. However, inorganic soil nitrogen was also highly linearly correlated with both MAP ([N] = 0.19 MAP – 32, r²= 0.96, p= 0.003) and ANPP (ANPP = 2.6 [N_inorganic]+59.4, r²= 0.79, p= 0.042), suggesting a direct control of precipitation on nitrogen turnover and an interaction with nitrogen availability in controlling carbon gain. The asynchrony of precipitation and changes in dominant vegetation may play important roles in determining the carbon‐nitrogen interactions along this rainfall gradient.     

Integrating sub-lethal heating with Brassica amendments and summer irrigation for control of Macrophomina phaseolina

Effects of varying intensities of sub-lethal heating were ascertained in improving the efficiency of Brassica amendments and summer irrigation on survival of Macrophomina phaseolina, a dry root rot pathogen. Sub-lethal heating (45–55°C) of M. phaseolina infested dry soil reduced the viable propagules by only 12.8% in a period of 90 days. One summer irrigation without sub-lethal heating caused 33.9% reduction in M. phaseolinapropagules, which improved to 43.3% when it was combined with 60 days of sub-lethal heating. Addition of the Brassicaamendments to irrigated soil resulted in significant reduction (60.4–71.6%) in counts of M. phaseolinabut this reduction improved (89.4–96.1%) when sub-lethal heating was combined with amendments. Mustard oil-cake (0.18% w/w) was found to be the most effective with reduction but a 94% inoculum reduction by mustard pod straw (0.36% w/w) was also achieved at 0–30 cm soil depth under similar conditions. Moderate heat level could not exert detectable weakening effect on M. phaseolinapropagules. These results suggest a practical cultural control of soil-borne pathogens by combining sub-lethal heating, Brassica amendments with one summer irrigation.     

Tillage effect on seasonal nitrogen availability in corn supplied with legume green manures

Colonization of sorghum by Macrophomina phaseolina in field plots was determined at nitrogen fertilization rates of 0, 56, 112, and 168 kg ha^-1 in 1988 and 0, 84, 168, and 256 kg ha^-1 in 1989. Above ground plant tissue and roots were sampled monthly to determine total nitrogen and percent colonization of root segments by natural inoculum. Root infection was not affected by nitrogen treatment, but was affected by growth stage and environment. High root infection occurred before reproductive development (growth stage 3) in 1988 and was associated with hot, dry weather early in the growing season. In 1989, when the weather was cool and wet, root infection began after reproductive development (growth stage 4). The effect of nitrogen treatments on lesion length was determined in sorghum stalks artificially inoculated with M. phaseolina. Lesion lengths were significantly affected by both nitrogen treatments and growth stage. Lesions were significantly longer with all nitrogen treatments at growth stage 9 than with the no-nitrogen treatment, and lesions tended to increase with increased levels of nitrogen fertilization. Significant increases in lesion length occurred between growth stages 5, 7, and 9 in 1988 and between 7 and 9 in 1989. This study demonstrates that nitrogen fertilization affects colonization of sorghum stalks but not root infection by M. phaseolina.     

Interrelationships Among Macrophomina phaseolina,Criconemella xenoplax,and Tylenchorhynchus annulatus on Grain Sorghum

Microplot experiments were established in 1992, 1993, and 1994 to investigate the relationships among Macrophomina phaseolina, Criconemella xenoplax, mad Tylenchorhynchus annulatus on grain sorghum in Louisiana. A factorial treatment arrangement of two grain sorghum hybrids (De Kalb DK 50 and Pioneer hybrid 8333), three levels of M. phaseolina (0, 10, and 100 colony-forming units (CFU)/g soil), and three nematode inoculum levels (0, 1x, and 2x) were used. Nematode inocula at 1x levels were 929, 1,139, and 1,445 C. xenoplax and T. annulatus/microplot in 1992, 1993, and 1994, respectively. Plants were harvested after 90-105 days. In all 3 years, grain sorghum root and head dry weights were suppressed as nematode inoculum level increased. These reductions were detected both in the absence and in the presence of M. phaseolina at 10 CFU/g. Reproduction of both nematode species was suppressed by M. phaseolina. Interactions between M. phaseolina and nematodes were antagonistic with regard to plant dry weights, yield, and nematode reproduction, so that combined effects were less than the sum of the effect of each pathogen alone.     

Streptomyces sp. S160: a potential antagonist against chickpea charcoal root rot caused by Macrophomina phaseolina (Tassi) Goid

Biological control of charcoal root rot disease caused by Macrophomina phaseolina in chickpea was studied by using Streptomyces sp. S160. This biocontrol agent (BCA) inhibited the mycelial growth of M. phaseolina by 50 % in vitro and significantly reduced charcoal rot incidence in the greenhouse by 33.3 %. The greenhouse experiment revealed that seed treatment along with soil application supported the highest germination (88.6 %), vigor index (7326.91) and reduced root rot incidence (12.5 %) in comparison to seed treatment and soil application alone. BCA enhanced the growth and helped in inducing resistance against charcoal rot disease of chickpea caused by M. phaseolina by increasing activity of defense-related enzymes in chickpea plants, leading to the synthesis of defense chemicals in plants. BCA (Streptomyces sp. S160) was also characterized and identified by using polyphasic approaches including 16S rDNA sequencing.     

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.