Proteo-metabolomic Dissection of Extracellular Matrix Reveals Alterations in Cell Wall Integrity and Calcium Signaling Governs Wall-Associated Susceptibility during Stem Rot Disease in Jute.

The plant surveillance system confers specificity to disease and immune states by activating distinct molecular pathways linked to cellular functionality. The extracellular matrix (ECM), a preformed passive barrier, is dynamically remodeled at sites of interaction with pathogenic microbes. Stem rot, caused by Macrophomina phaseolina, adversely affects fiber production in jute. However, how wall related susceptibility affects the ECM proteome and metabolome remains undetermined in bast fiber crops. Here, stem rot responsive quantitative temporal ECM proteome and metabolome were developed in jute upon M. phaseolina infection. Morpho-histological examination revealed that leaf shredding was accompanied by reactive oxygen species production in patho-stressed jute. Electron microscopy showed disease progression and ECM architecture remodeling due to necrosis in the later phase of fungal attack. Using isobaric tags for relative and absolute quantitative proteomics and liquid chromatography-tandem mass spectrometry, we identified 415 disease-responsive proteins involved in wall integrity, acidification, proteostasis, hydration, and redox homeostasis. The disease-related correlation network identified functional hubs centered on α-galactosidase, pectinesterase, and thaumatin. Gas chromatography-mass spectrometry analysis pointed toward enrichment of disease-responsive metabolites associated with the glutathione pathway, TCA cycle, and cutin, suberin, and wax metabolism. Data demonstrated that wall-degrading enzymes, structural carbohydrates, and calcium signaling govern rot responsive wall-susceptibility. Proteomics data were deposited in Pride (PXD046937; PXD046939).

GC-MS analysis and antifungal potential of flower extract of Acacia nilotica subsp. Indica against Macrophomina phaseolina.

Macrophomina phaseolina is a wide host ranged soil-borne fungal plant pathogen. It infects more than 500 host plant species belonging to 100 families. Many important oil-seed and leguminous crops are known to be attacked by this devastating plant pathogen. In the present study, antifungal potential of flowers of a leguminous tree Acacia nilotica subsp. indica, was assessed against this pathogen through bioassays guided fractionation. Initially, methanolic extracts of 1 %-5 % of leaf, flower, root-bark and stem-bark of the plant species under consideration were evaluated for their antifungal potential against the target pathogen. Among these, the best antifungal activity was shown by flower extract. The reduction in growth of the test fungal strain was 27-49 %, 4-40 % and 2-27 % due to flower, root-bark and leaf extracts, respectivey, over control. Flower extract was partitioned using n-hexane, chloroform, ethyl acetate and n-butanol as the solvents. Bioassays guided study of these fractions of methanolic extract of flower revealed that high antifungal potential was shown by n-hexane and chloroform fractions against M. phaseolina causing 26-53 % and 28-50 % decline in fungal biomass, respectively, as compared to that of control. GC-MS analysis of chloroform fraction revealed the presence of 27 compounds in this fraction. Among these cyclopentanol,-1-methyl (10.93 %) was the predominant compound followed by methyl, 4,4-dimethyl butanoate (7.04 %), 1-pentanol (6.80 %), 2-propanol, 1-cyclopropyl (6.11 %), 1H,imidazole-4-5-dihydro-2-methyl (5.93 %), trichloroethane (5.91 %), carbonic acid-ethyl hexyl ester (4.59 %), 1,4-butandiol,2,3-bis(methylene)- (4.54 %) and [S]-3,4-dimethyl pentanol (4.48 %).

Tuber pathogenicity of Macrophomina phaseolina strain 3 a isolated from rotten cassava tuber from farm lands in Osogbo, Osun State, Nigeria, its virulence genes and ADMET properties.

BACKGROUND: Macrophomina phaseolina is a pathogen that causes an opportunistic disease that spreads by soil and seeds and affects more than 500 different plant species, like fruits, trees, and row crops. Mycotoxins, such as phaseolinic acid, and phaseolinone, are produced by M. phaseolina isolates in previous investigations; however, the production of these mycotoxins seems to vary depending on the host and the region. METHODS AND RESULTS: In this study, Macrophomina phaseolina strain 3 A was isolated from rotten cassava tuber and identified using the analysis of the sequences of the internal transcribed spacer region. The isolate was inoculated on a fresh healthy cassava tuber at 25 °C and tuber-rotting potential was monitored for 4 weeks. Virulence genes MPH_06603, MPH_06955, and MPH_01521 were determined with designed primers, and secondary metabolites were characterized by FTIR and GCMS. The rotten tuber effect was observed from the 2nd week of the experiment with severe tuber rot and weight reduction. The PCR showed the presence of MPH_06603 virulence gene. The GCMS showed N-Methylpivalamide (115.0 m/z), Butane, 1,4-dimethoxy- (119.0 m/z), and 5-Hydroxymethylfurfural (126.0 m/z) were the predominant metabolites produced by the pathogen. The compounds in the metabolites inhibit CYP3A4 enzymes, cause eye irritation, and Human Ether-a-go-go-related gene inhibition. CONCLUSION: This study revealed that M. phaseolina was responsible for the cassava tuber rot which leads to a lower yield of farm produce. The metabolites produced are toxic and unsafe for human consumption. It is suggested that farmers should destroy any cassava affected by this pathogen to prevent its toxic effects on humans and animals.

Exaggerated Plurivory of Macrophomina phaseolina: Accounting for the Large Host Range Claim and the Shifting of Scientific Language.

Macrophomina phaseolina is a plant pathogenic fungus that is frequently described as having a broad host range encompassing more than 500 species. We noticed that citations provided in support of this statement do not actually demonstrate such a broad host range. To elucidate the true documented host range of this fungus, we initiated a literature meta-analysis of 894 publications on M. phaseolina since 1913. We discovered that the first host range summaries did not require Koch's postulates or other experimental demonstrations of pathogenicity. Most of the available early host claims were based on tenuous associations between the fungus and symptoms, sometimes without reporting isolation or morphological examination in vitro. These statements apparently led to a pattern of increasingly exaggerated host range claims, without support from a primary reference, until the claim that M. phaseolina has 500 hosts became common in the early 2000s. At present, the scientific community typically requires Koch's postulates to characterize pathogenicity on a new host. Among all the available literature, we only found primary experimental evidence for M. phaseolina's pathogenicity on 97 hosts; 74 hosts confirmed by Koch's postulates and 23 hosts with all steps from Koch's postulates completed except for recovery of the pathogen from symptomatic tissues. This study demonstrates how scientific concepts can change over time and necessitate changes to historic axioms. We propose that the hyperbole surrounding the host range of M. phaseolina has obscured an accurate depiction of its biology.

First Report of Charcoal Rot on Tobacco Caused by Macrophomina phaseolina in Hunan Province, China.

Flue-cured tobacco (Nicotiana tabacum L.) is a significant cash crop globally. In August 2022, necrotic lesions on stem associated with root rot and wilting were observed on flue-cured tobacco (Cv. Yunyan 87) in fields located in Banxin village (27.95N,109.60E) of Fenghuang county in Xiangxi Autonomous Prefecture, Hunan Province, China. The affected and damaged area of tobacco is approximately 10 hectares, with adisease incidence of 60%. Lots of small black speckling within the lower stem of the affected plant, vascular tissue changed to black, dry rot, and looked like charcoal breezes. Small pieces were cut from healthy and diseased tissues, surface sterilized with 5% NaClO for 3 min and 75% ethanol for 1 min, rinsed with sterile distilled water and air-drying, incubated on oat medium incubated at 28℃ for five days. These isolates grew fast and produced typical black microsclerotia. The morphological were septate hyphae and microsclerotia. The microsclerotia were black and regularly round, with a 42.5 - 92.9 µm diameter. These morphological features were consistent with Macrophomina phaseolina (Smith and Wyllie 1999). The internal transcribed spacer (ITS) rDNA and translation elongation factor 1-α (TEF1-α) genes of three representative isolates were amplified and sequenced using the primers ITS1/ITS4 and EF1-728F/EF2R (Machado et al. 2019). Our resulting sequences (GenBank accessions OR435093, OR435101, OR435102 for ITS; OR891780, OR891781 and OR891782 for EF1-α) showed 99-100% similarity with M. phaseolina by NCBI blast. Phylogenetic analysis was conducted using MEGA-X software with the NJ method. The combined sequences grouped with isolates to M. phaseolina with 100% bootstrap support. The strain XF22 has been sent to the China General Microbiological Culture Collection Center (CGMCC3.25349). Pathogenicity tests were conducted by inoculating potted plants (six plants per isolate, three times) from 45 day-old tobacco seedlings cv. Yunyan 87. Stems were randomly gently scratched with sterile needles, and a 5 mm agar disc with mycelium of the pathogen was attached to the surface of each wound, with a sterilized agar disc as control. Inoculated seedlings were incubated in growth chambers at 26℃ and 60% RH with a 12 h photoperiod/day. After ten days, symptoms that brown or black lesions on the inoculated lesions were dotted with numerous black, hard microsclerotia similar to those naturally occurring on the diseased plants, but not on the control plants. The same pathogen was re-isolated consistently, fulfilling Koch's postulates. Based on morphological, molecular, and pathogenicity test results, these isolates were identified as M. phaseolina. Charcoal rot of tobacco, caused by M. phaseolina was previously found in Guangxi in 1989 (Zhu et al. 2002), while this is the first report of M. phaseolina causing charcoal rot on flue-cured tobacco in Hunan, China. We speculate that the planting area is influenced by the preceding crop sesame. The soil carries M. phaseolina, which can cause stem rot of sesame, leading to the occurrence of tobacco charcoal rot. Our results indicated that charcoal rot caused by M. phaseolina is a new threat to flue-cured tobacco production and lue-cured tobacco might be acting as a reservoir and spreading this pathogen to other economically crops in China.

Effect of drought stress during seed development on charcoal rot and yield of soybean.

The primary controls for charcoal rot in soybean, caused by the fungal pathogen Macrophomina phaseolina, are to avoid drought stress and to plant a moderately resistant cultivar. The effects of irrigation and cultivar were determined in 2011 and 2013 at the Lon Mann Cotton Research Station, Marianna, AR. Four soybean cultivars (Hutcheson, Osage, Ozark, and R01581F), were planted in plots with or without added M. phaseolina inoculum and subjected to three furrow irrigation regimes: full season irrigation (Full), irrigation terminated at R5 (CutR5), and non-irrigated (NonIrr). Normalized difference vegetative index (NDVI) was measured at R3 and R6. At harvest, plants and yields were collected. Roots and stems were split and the extent of visible colonization by M. phaseolina microsclerotia was assessed in the roots with a 1-5 scale (RSS) and the percent plant height stem discoloration (PHSD) measured. Precipitation in September and October was 54 and 65% below the 30-year average in 2011 and 2013, respectively. The CutR5 irrigation treatment resulted in one less irrigation than Full each year, but CutR5 NDVI's at R6 and yields were significantly lower than those with Full and not significantly different than those of NonIrr. The CutR5 RSS ratings were greater than either Full or NonIrr. Plant colonization by M. phaseolina was negatively correlated to yield in 2011 but not in 2013. No premature plant death caused by charcoal rot was observed in either year. These results indicated that late season drought stress may be more important to charcoal rot development than drought stress throughout the season, but other factors are needed to trigger early plant death and subsequent yield losses observed in grower fields.

First report of Macrophomina phaseolina causing сharcoal rot in soybean (Glycine max (L.) Merr.) plants in Uzbekistan.

The soybean production area is expanding in Uzbekistan. Soybeans were planted on an area of 10 thsd ha and the harvest amounted to 30 thsd metric tons in 2023 (IPAD, https://ipad.fas.usda.gov/countrysummary). Macrophomina phaseolina (Mp) is a soil- and seed-borne fungal pathogen causing economically important diseases of legume crops (Pennerman et al. 2024). Drought stress and a warm climate are favorable to this pathogen (Irulappan et al. 2022). Under these conditions, its microsclerotia survive for a longer period and become more virulent (Chamorro et al. 2015). In August 2022, typical symptoms of charcoal rot were observed in about 25% of "Orzu" soybean cultivar affecting 6 ha located on the experimental base "Durmon" of our institute. Diseased plants displayed the following charcoal rot symptoms: leaves turn yellow, then wilt, die, and remain attached to the plant; the lower portion of the stem and tap root have a light gray or ashy black discoloration; tiny black specks on the lower stem and root; after splitting the stem, it has the appearance of fine charcoal powder. In order to determine the causal agent of these symptoms, a total of 17 diseased plants were collected from focal lesions in soybean plantings. From each plant, twelve sections of stem and root tissue were selected, cut into small 5-mm pieces, and surface sterilized with 1% sodium hypochlorite for four minutes, then rinsed three times with sterile distilled water. The disinfected tissues were dried on sterile filter paper for 5 min and placed on PDA Petri plates, which were incubated in an incubation chamber for 3 days (16 h light (26oC) and 8 h dark (18oC)). Fungi were subsequently subcultured on PDA and incubated for 7 days to obtain pure cultures. Six monohyphal colonies were purified. The colonies showed dense growth, with a gray initial mycelium becoming darker with aging. After 8 days on PDA, black-colored microsclerotia with spherical to oblong shapes were observed. On average, they measured 60 µm in width and 130 µm in length (n = 30). From six isolated monohyphal colonies, one has been chosen for molecular-genetic identification. Molecular-genetic analysis was conducted by amplification and sequencing of the ITS region with the ITS1 and ITS4 primers (White et al. 1990). The resulting sequence was deposited in the NCBI database under accession number OQ073450. After BLAST analysis (Altschul et al. 1990) it was 100% identical with the reference sequences of Mp (accession MT039671, MT039663 and MH496040) isolated in sugar beet, maize and sunflower, respectively, from Serbia. In order to verify the pathogenicity, soybean seedlings (cv. Orzu) were dipped into spore suspension (1 × 107 spores/ml) of sequenced strain R-17 for 1 minute and transferred to a 15 cm diameter plastic pot with 350 g of sterilized soil mix. After 25 days, the inoculated plants showed classic charcoal rot symptoms, while the control plants remained healthy. The pathogen was successfully reisolated from the infected seedlings onto PDA, fulfilling Koch's postulate. The identity of the re-isolated strain was confirmed by morphological features and sequencing of the ITS region. It should be noted that in Uzbekistan, Mp has not been documented in any plants. Therefore, according to our knowledge, this is the first report of this fungus affecting soybean plants in Uzbekistan. Since molecular-genetic analysis of the R-17 strain showed clustering with strains from Serbia, we speculate that there may have been a recent introduction of Mp from Serbia into Uzbekistan. This assumption is additionally confirmed by the fact that Serbia is the largest seed exporter in Uzbekistan. The increase in charcoal rot disease poses a major challenge to soybean production in Uzbekistan. Understanding the genetic diversity of Mp can be utilized to manage this disease, improve soybean yield, and help soybean breeding programs in Uzbekistan.

Cover Crop and Crop Rotation Effects on Tissue and Soil Population Dynamics of Macrophomina phaseolina and Yield Under No-Till System.

The effects of crop rotation and winter cover crops on soybean yield and colony-forming (CFU) units of Macrophomina phaseolina, the causal agent of charcoal rot (CR), are poorly understood. A field trial was conducted from 2011 to 2015 to evaluate (i) the impact of crop rotation consisting of soybean (Glycine max [L.] Merr.) following cotton (Gossypium hirsutum L.), soybean following corn (Zea mays L.), and soybean following soybean over a 2-year rotation and its interaction with cover crop and (ii) the impact of different cover crops on a continuous soybean crop over a 5-year period. This trial was conducted in a field with 10 subsequent years of cover crop and rotation treatments. Cover crops consisted of winter wheat (Triticum aestivum L.) and Austrian winter pea (Pisum sativum L. subsp. sativum var. arvense), hairy vetch (Vicia villosa Roth), and a fallow treatment was evaluated with and without poultry litter application (bio-cover). Tissue CFU of M. phaseolina varied significantly between crop rotation treatments: plots where soybean was grown following cotton had significantly greater tissue CFU than plots following soybean. Poultry litter and hairy vetch cover cropping caused increased tissue CFU, though this effect differed by year and crop rotation treatment. Soil CFU in 2015 was substantially lower compared with 2011. However, under some crop rotation sequences, plots in the fallow treatment had significantly greater soil CFU than plots where hairy vetch and wheat was grown as a cover crop. Yield was greater in 2015 compared with 2011. There was a significant interaction of the previous crop in the rotation with year, and greater yield was observed in plots planted following cotton in the rotation in 2015 but not in 2011. The result from the continuous soybean planted over 5 years showed that there were no significant overall effects of any of the cover crop treatments nor was there interaction between cover crop treatment and year on yield. The lack of significant interaction between crop rotation and cover crop and the absence of significant differences between cover crop treatments in continuous soybean planting suggest that cover crop recommendations for midsouthern soybean growers may need to be independent of crop rotation and be based on long-term crop needs.

Genome-Wide Association Study Reveals a QTL Region for Ashy Stem Blight Resistance in PRA154 Andean Common Bean.

Ashy stem blight (ASB) caused by Macrophomina phaseolina (Tassi) Goidanich affects the common bean (Phaseolus vulgaris L.) at all growing stages. Higher levels of resistance were observed in Andean common beans, but specific resistant quantitative trait loci (QTLs) conferring resistance to this pathogen have not been reported in this gene pool. The objectives of this research were to: (i) conduct a genome-wide association study (GWAS) and QTL mapping for resistance in the Andean breeding line PRA154; and (ii) identify single nucleotide polymorphism (SNP) markers and candidate genes for ASB resistance. Phenotyping was conducted under greenhouse conditions by inoculating the 107 F6:7 recombinant inbred lines (RILs) derived from the cross between the susceptible cultivar 'Verano' and the partial-resistant breeding line PRA154 twice with the M. phaseolina isolate PRI21. Genotyping was performed with 109,040 SNPs distributed across all 11 P. vulgaris chromosomes. A novel major QTL was located between 28,761,668 and 31,263,845 bp, extending 2.5 Mbp on chromosome Pv07, and the highest significant SNP markers were Chr07_28761668_A_G, Chr07_29131720_G_A, and Chr07_31263845_C_T with the highest LOD (more than 10 in most of the cases) and R-squared values, explaining 40% of the phenotypic variance of the PRI21 isolate. The gene Phvul.007G173900 (methylcrotonyl-CoA carboxylase alpha chain and mitochondrial 3-methylcrotonyl-CoA carboxylase 1 [MCCA]) with a size of 10,891 bp, located between 29,131,591 and 29,142,481 bp on Pv07, was identified as one candidate for ASB resistance in PRA154, and it contained Chr07_29131720_G_A. The QTL and genetic marker information could be used to assist common bean breeders to develop germplasm and cultivars with ASB resistance through molecular breeding.

Pioneering Nit Gene Exploitation to Develop Molecular Diagnostic Assay for Rapid Detection of Cotton Root Rot Incitant, Macrophomina phaseolina (Tassi) Goid, in Field Soil.

Cotton root rot caused by Macrophomina phaseolina pose a significant threat to cotton production, leading to substantial yield and quality losses. Early and accurate diagnosis of this pathogen in soil is crucial for effective disease management. This study presents a pioneering investigation into the utilization of the nit gene encoding nitrilase for the development of a molecular diagnostic assay aimed at the rapid detection of M. phaseolina in field soils. The methodology involved the design and validation of primers targeting the Nit gene sequence, followed by the optimization of PCR conditions for efficient amplification. Leveraging state-of-the-art molecular techniques, the assay offers a novel protocol to accurately identify the presence of M. phaseolina in soil with high sensitivity and specificity. The specificity of the designed primers was confirmed through PCR amplification using DNA from M. phaseolina and other related fungi. Sensitivity tests demonstrated that the PCR assay reliably detected M. phaseolina DNA at concentrations as low as 1 ng. Furthermore, the performance of the diagnostic assay was rigorously evaluated using field soil samples with a known status of M. phaseolina infection, demonstrating its reliability and efficacy in real-world scenarios. This study introduces a novel molecular marker for the detection of M. phaseolina and offers a rapid and efficient means for screening M. phaseolina in large soil samples with minimal time and manpower.

Genome-wide characterization of the wall-associated kinase-like (WAKL) family in sesame (Sesamum indicum) identifies a SiWAKL6 gene involved in resistance to Macrophomina Phaseolina.

BACKGROUND: Sesame charcoal rot caused by Macrophomina phaseolina is one of the most serious fungal diseases in sesame production, and threatens the yield and quality of sesame. WAKL genes are important in the plant response to biotic stresses by sensing and transmitting external signals to the intracellular receptor. However, there is still a lack about the WAKL gene family and its function in sesame resistance to M. phaseolina. The aim of this study was to interpret the roles of WAKL genes in sesame resistance to M. phaseolina. RESULTS: In this study, a comprehensive study of the WAKL gene family was conducted and 31 WAKL genes were identified in the sesame genome. Tandem duplication events were the main factor in expansion of the SiWAKL gene family. Phylogenetic analysis showed that the sesame SiWAKL gene family was divided into 4 groups. SiWAKL genes exhibited different expression patterns in diverse tissues. Under M. phaseolina stress, most SiWAKL genes were significantly induced. Notably, SiWAKL6 was strongly induced in the resistant variety "Zhengzhi 13". Functional analysis showed that SiWAKL6 was induced by salicylic acid but not methyl jasmonate in sesame. Overexpression of SiWAKL6 in transgenic Arabidopsis thaliana plants enhanced their resistance to M. phaseolina by inducing the expression of genes involved in the salicylic acid signaling pathway and reconstructing reactive oxygen species homeostasis. CONCLUSIONS: Taken together, the results provide a better understanding of functions about SiWAKL gene family and suggest that manipulation of these SiWAKL genes can improve plant resistance to M. phaseolina. The findings contributed to further understanding of functions of SiWAKL genes in plant immunity.

MicroRNA397 regulates tolerance to drought and fungal infection by regulating lignin deposition in chickpea root.

Plants deposit lignin in the secondary cell wall as a common response to drought and pathogen attacks. Cell wall localised multicopper oxidase family enzymes LACCASES (LACs) catalyse the formation of monolignol radicals and facilitate lignin formation. We show an upregulation of the expression of several LAC genes and a downregulation of microRNA397 (CamiR397) in response to natural drought in chickpea roots. CamiR397 was found to target LAC4 and LAC17L out of twenty annotated LACs in chickpea. CamiR397 and its target genes are expressed in the root. Overexpression of CamiR397 reduced expression of LAC4 and LAC17L and lignin deposition in chickpea root xylem causing reduction in xylem wall thickness. Downregulation of CamiR397 activity by expressing a short tandem target mimic (STTM397) construct increased root lignin deposition in chickpea. CamiR397-overexpressing and STTM397 chickpea lines showed sensitivity and tolerance, respectively, towards natural drought. Infection with a fungal pathogen Macrophomina phaseolina, responsible for dry root rot (DRR) disease in chickpea, induced local lignin deposition and LAC gene expression. CamiR397-overexpressing and STTM397 chickpea lines showed more sensitivity and tolerance, respectively, to DRR. Our results demonstrated the regulatory role of CamiR397 in root lignification during drought and DRR in an agriculturally important crop chickpea.

Biochemical and molecular depictions to develop ech42 gene-specific SCAR markers for recognition of chitinolytic Trichoderma inhibiting Macrophomina phaseolina (Maubl.) Ashby.

Trichoderma isolates were inhibited variably in-vitro growth of soil-borne phytopathogen Macrophomina phaseolina (Maubl.) Ashby causes root rot in cotton. The growth inhibition of test-pathogen was found to be higher (90.36%) in T. viride NBAIITv23 followed by T. koningii MTCC796 (85.77%) under dual culture antagonism. The microscopic examination suggested that the antagonists Tv23 and MTCC796 adopted mycoparasitism as a strong mode of action to restrain pathogen growth. However, antagonists T. harzianum NBAIITh1 (77.89%) and T. virens NBAIITvs12 (61.74%) demonstrated strong antibiosis action for growth inhibition of the test pathogen. A significant positive correlation was established between the growth inhibition of M. phaseolina and the release of cell wall degrading enzymes- chitinase (p = 0.001), ß-1,3, glucanase (p = 0.01), and protease (p = 0.05) under the influence of pathogen cell wall. The chitinase and ß-1,3, glucanase activities were elevated 2.09 and 1.75 folds, respectively, in potent mycoparasitic Tv23 strain influenced by a pathogen cell wall compared to glucose as a carbon source. The three unique DNA-RAPD fragments OPA-07(1033), OPA-16(983), and OPO-15(239), amplified by potent mycoparasitic Tv23 strain, were subjected to DNA sequencing and derived functional 864 bp from OPA-16(983) and have sequence homology to ech42 gene with partial CDs of 262 amino acids (nucleotide accession No. KF723016.1 and protein accession No.AHF57046.1). Novel SCAR markers were developed from a functional sequence of OPA-16 fragments and validated across the genomic DNA of eleven Trichoderma antagonists. The novel SCAR markers evolved from the RAPD-SCAR interface to authenticate chitinolytic Trichoderma associated with mycoparasitic action for eco-friendly biocontrol activity.

First Report of Macrophomina phaseolina Causing Leaf Blight of Cassava in China.

Cassava (Manihot esculenta Crantz) is an important tropical and subtropical crop that feeds nearly 600 million people worldwide and is widely grown in Hainan Province, China (Vanderschuren et al., 2014). In November 2021, leaf blight symptoms were observed on South China 6 (SC6) cassava plants in Haikou City, Hainan Province, China. The disease was presented in almost every cassava plant we observed. The rotten leaves were shown to be infected but not the root or stem. The lesions started on the plant's lower leaves and gradually developed on the upper leaves of the entire cassava plant. The infected leaves gradually withered. Microscopic observation showed that the infected leaves exhibited necrotic lesions with pycnidial structures all over their surface. Diseased leaf segments (4 × 4 mm) were disinfected for 30 seconds with sodium hypochlorite 1% solution and then rinsed with sterile water for 30 seconds before being placed on potato dextrose agar (PDA) medium. Plates were incubated at 28°C in complete darkness. Marginal hyphae were picked and placed on a new PDA medium, and pure cultures were obtained after multiple transfers. The hyphae started white and gradually changed to a fluffy black-gray color as it grew on the PDA. Microscopic observation showed that there were a large number of ellipsoidal microsclerotia between the hyphae. Microsclerotia were sub fusiform, and hyaline, with a length of about 40 µm. The ribosomal internal transcribed spacer (ITS) region, ribosomal small subunit (SSU) region, and ribosomal large subunit (LSU) region of the isolate were amplified and sequenced using primers ITS1 and ITS4, NS1 and NS4 (White et al., 1990), and LROR and LR5 (Moriya et al., 2005), respectively. The obtained ITS (GenBank accession no. OP185242), SSU (GenBank accession no. OQ165195), and LSU (GenBank accession no. OQ118350.1) had 99.8% (100% coverage), 100% (100% coverage), and 100% (100% coverage) identities with the references ITS (GenBank accession no. KF951698), SSU (GenBank accession no. KF766281.1), and LSU (GenBank accession no. KF766364.1) in Macrophomina phaseolina, respectively. A phylogenetic tree was constructed with software MEGA7 using the maximum likelihood method, showing that the isolate was grouped in the same clade as M. phaseolina. To prove Koch's postulates, five healthy SC6 cassava plants (2-month-old) with 4-6 leaves were wounded with a small pin and inoculated with PDA blocks (3 × 3 mm) excised from the margin of a 7-day-cultured colony (Hu et al., 2022). Healthy plants treated with sterile PDA plugs served as controls. All plants were grown at 25°C with a 12-h light/dark rotation. After 7 days, typical blight symptoms developed on leaves inoculated with M. phaseolina, but not on the controls. The fungus was isolated from infected leaves. Based on molecular identification, M. phaseolina was re-isolated from leaves with leaf blight symptoms. Macrophomina is typically found to cause root and lower stem rot on cassava in Africa (Msikita et al., 1998). To the best of our knowledge, this is the first report of M. phaseolina causing leaf blight on cassava in China. Our finding provides a foundation to management of this disease.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Hemp in Croatia.

The production of industrial hemp (Cannabis sativa) has had a sharp increase in the past five years in Croatia (Mihelcic 2017). Production has been in constant increase, from 1560 ha in 2015 to 2476 ha in 2019 (PAAFRD). In August 2019, numerous (~1.5%) premature wilted hemp plants (cultivar Fibranova) were observed in commercial fields in Vladislavci (45.4646950° N, 18.5674770° E), around Osijek, Croatia. Diseased plants showed symptoms of chlorosis, rapidly wilting, necrosis and prematurely dying. The stalk of diseased plants was completely desiccated, while internal tissues were spongy and fluffy. Near the soil line, discoloration of the stalk with small spherical to oblong black microsclerotia was detected. Roots were necrotic with brown-gray areas. Twenty root and crown segments of the infected plants (2- to 3-mm long pieces) were surface sterilized with 2% NaOCl for 5 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar (PDA, pH 6.2) media, containing 250 mg per liter of chloramphenicol to inhibit bacterial growth (Zveibil and Freeman 2005). The culture plates were incubated at 28 ± 2°C for 7 days in the dark and purified to obtain a pure culture that produces numerous, dark, hard, ovoidal-shaped sclerotia, averaging 140 x 52 µm (n=50). The single sclerotium isolate produced flat light to dark gray colonies with entire margins. Based on field symptoms, colony and microsclerotia morphology, the fungus was identified as Macrophomina phaseolina (Tassi) Goid (Marquez et al. 2021). Total DNA from the isolate was extracted with Extract-N-Amp Plant PCR Kit (Sigma-Aldrich Co., USA). To confirm morphological identification, part of the TEF 1-alpha gene region was amplified using EF1-728F (Carbone and Kohn 1999) and EF2 (O'Donell et al. 1998). The sequence of the isolate MP1 (212 bp - GenBank accession no. OQ389757), showed 100% nucleotide sequence identity to the reference sequence of M. phaseolina GenBank sequence MG434668 (Casano et al. 2018). Eighteen hemp plants (cv. Fibranova) were sown in six plastic pots (three hemp plants per pot) for the pathogenicity test. Ten-day-old M. phaseolina culture (isolate MP1) was used for inoculum preparation. Each pot of one-week-old plant was irrigated with 100 ml of a microsclerotia suspension (105 microsclerotia/ml)(Abied et al. 2018). Plants were held at 28°C and 70% relative humidity in a growth chamber (aralab, Fitoclima 10.000 HP) with a 16-hour photoperiod. Pots with control plants were irrigated with the same amount of sterile distilled water. Ten weeks after inoculation percentage of wilted plants was 77.78%. In the control variant all plants were healthy. M. phaseolina was reisolated from inoculated plants and morphologically identified. With the liberalization of the law, hemp production in Croatia is increasing, which could result in general disease problems and the disease caused by M. phaseolina. Charcoal rot will be expressed in years with dry and warm summers (Lodha and Mawar 2020), and relatively short, cool, rainy winters (Nevo et al. 2012), which has become common in the last decade in Croatia. To our knowledge, this is the first report of M. phaseolina on hemp in Croatia. The authors declare no conflict of interest.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Dry Bean (Phaseolus vulgaris L.) in Western Canada.

In 2021, several dry bean (Phaseolus vulgaris L.) plants at the mid-seed-fill growth stage displaying wilting, chlorosis of the leaves and reduced vigor were collected near the Pembina - Emerson Border of Manitoba, Canada and North Dakota, USA. When symptomatic plants were examined, gray to dark brown discoloration was observed on the lower stem and the roots. Afterwards, brown to black discoloration was noticed on stem and root sections. Root and lower stem pieces (1 to 2 cm) from affected plants were surface sterilized with 70 % ethanol, followed by 1% NaOCl, rinsed twice in sterilized water, air dried on sterilized filter papers, and placed on potato dextrose agar (PDA) amended with 1 mg/mL of streptomycin sulfate. The PDA plates were incubated at 28°C with 12 h light/12 h dark for 10 days. The growing hyphae were transferred using the hyphal tip method to new PDA plates. Growing cultures were initially hyaline and turned from light gray to dark brown or black with age. Abundant dark and spherical to oblong shaped sclerotia with an average diameter of 97.9 µm (range: 66.8 to 143.5 µm, n =30) formed on the pure cultures 7 days after incubation. Additional pure culture was obtained through an isolation of a single microsclerotium followed by a single hyphal tip transfer. One isolate was identified as Macrophomina phaseolina based on morphological characteristics (Smith and Wyllie 1999). The morphological identity was confirmed by sequencing the rDNA internal transcribed spacer (ITS) region with universal primers ITS1/ITS4 (White et al. 1990) and calmodulin (CAL) and translation elongation factor-1 alpha (TEF-1α) genes with MpTefF/MpTefR, and MpCalF/MpCalR primer sets (Santos et al. 2020), respectively. The online resource Basic Local Alignment Search Tool (BLAST; https://www.ncbi.nlm.nih.gov/BLAST) confirmed the fungus identity as 100% M. phaseolina. The sequences of the original isolate BF21-25 were deposited in GenBank with accession numbers OQ615297 (ITS), OR357630 (CAL), and OR363106 (TEF-1α). To confirm pathogenicity, bioassays were conducted under controlled conditions. Four seeds of cultivar 'Etna' were sown per pot, and five pots were used for inoculated (approx. 4 × 105 microsclerotia/pot) and control (mock-inoculated with sterile PDA medium) treatments. For the inoculum, 20 g of macerated 10 to 14-day old M. phaseolina culture grown on PDA medium was applied to each pot using an inoculum layering technique. Pots were kept in the greenhouse with 28/17°C day/night, 13 h light/11 h dark cycle, and 70% relative humidity and watered weekly. Disease symptoms similar to those observed in the field were visible on all inoculated plants at the mid-seed-fill growth stage. Mock-inoculated control plants didn't show any symptoms. The experiment was repeated twice with similar results. The pathogen was re-isolated from infected plants to confirm Koch's postulates and identified as M. phaseolina based on the morphology and sequences of ITS, CAL and TEF-1α regions. To our knowledge, this is the first report of charcoal rot caused by M. phaseolina on dry bean in Western Canada.

First Report of Charcoal Rot on Soybean Caused by Macrophomina phaseolina in Manitoba, Canada.

Macrophomina phaseolina (Tassi) Goid. is a soilborne necrotrophic fungal pathogen causing charcoal rot on approximately 500 plant species worldwide (Mengistu et al. 2015). Charcoal rot occurs in eastern Canada and many regions of the USA, causing substantial yield losses in soybean [Glycine max (L.) Merr.] (Allen et al. 2017; Bradley et al. 2021; Wrather et al. 2001). However, it has not been reported in soybean in western Canada. Manitoba is the second largest soybean producer in Canada, comprising 31% of total seeded areas with 2.29 M acres in 2017 (Statistics Canada 2022). Still, soybean is a relatively new crop to Manitoba and annual surveys of soybean root diseases began in 2012. In August 2020, randomly selected soybean fields were surveyed for root diseases at 63 different locations in south-central and southwest Manitoba. A total of thirty diseased plants were sampled in a zigzag pattern at three random sites in each field and all samples were brought to the laboratory and rated for disease severity. All plants showed symptoms of root rot, and some samples exhibited wilting with yellowing-brown leaves attached to the stems by the petioles; when the taproot was sectioned longitudinally, black streaking could be observed. In the laboratory, 600 roots from 40 selected fields were processed for pathogen isolation and identification. A 1 cm section from each root was surface-sterilized in a 95% EtOH:5.25% NaOCl solution for 30 sec, rinsed in sterile water for 60 sec, and air-dried on sterilized filter paper in a laminar flow hood. Root tissues with two replicates were placed on potato dextrose agar (PDA) plates amended with streptomycin sulfate (2 mg/mL) and incubated at room temperature. Black microsclerotia were observed in cultures from three different fields and three individual fungal isolates were obtained from each field through isolation of a single microsclerotium and subsequent hyphal tip transfer. The mycelia were initially hyaline and turned gray to dark brown or black, forming numerous microsclerotia ranging in size from 13 to 61 µm long and 12 to 32 µm wide, based on measurements of approximately 100 microsclerotia per isolate using a Zeiss Axio Imager A2 microscope equipped with an AxioCam HRc (Carl Zeiss, Jena, Germany) and AxioVision software. The color of the microsclerotia was jet black and the shape was round to oblong or irregular, as described by Mengistu et al. (2015). Based on morphological characteristics and microscopic examination, three fungal isolates were identified as M. phaseolina (Mengistu et al. 2015). For molecular identification, genomic DNA was extracted from 10 to 14-day old mycelia and microsclerotia of each isolate using a ZymoBIOMICS™ DNA Miniprep Kit (Zymo Research Corp., Irvine, CA, USA) according to the manufacturer's instructions. The internal transcribed spacer (ITS) region, translation elongation factor-1α (TEF-1α), and calmodulin (CAL) genes were amplified using the primer sets ITS1/ITS4 (White et al. 1990), MpTefF/MpTefR, and MpCalF/MpCalR (Santos et al. 2020), respectively, according to the original reaction conditions. Subsequently, PCR products were sequenced at Eurofins Genomics (Louisville, KY, USA). BLASTn analysis in GenBank showed that the nucleotide sequences of these regions of the three isolates (NSRR20-MB-24, NSRR20-MB-34, and NSRR20-MB-40) matched multiple isolates of M. phaseolina with 100% query cover and 100% identity. Sequences were deposited in GenBank for the ITS (OK127887, OK142725, OK128266), TEF-1α (OR363103, OR363104, OR363105), and CAL (OR357627, OR357628, OR357629) regions. In addition, the ITS and TEF-1α sequences of the three novel isolates were further aligned with multiple previously reported isolates of M. phaseolina, M. pseudophaseolina, and M. euphorbiicola (Chen et al. 2013; Machado et al. 2019; Sarr et al. 2014) using Muscle and trimmed (Edgar 2004). Alignments were concatenated to generate a maximum likelihood tree. Once concatenated, sequences were re-aligned. The obtained alignments were employed to construct a phylogenetic tree using the max likelihood method and Tamura-Nei model (Tamura and Nei 1993) with 10,000 bootstrap replicates using MEGA 11 (Tamura et al. 2021). The ITS and TEF-1α analysis indicated that the isolates were grouped in three differentiated clades (Figure 1). Macrophomina phaseolina isolates clustered in the same clade at 98% similarity, with the three novel soybean isolates NSRR20-MB-24, NSRR20-MB-34, and NSRR20-MB-40 grouped closely in the cluster at 98% similarity and identified as M. phaseolina. In contrast, isolates of M. euphorbiicola formed another clade at 87% similarity and M. pseudophaseolina isolates grouped in a clade at 99%. The pathogenicity of the three isolates was evaluated under controlled conditions. Given that no information on charcoal rot resistance in soybean has been reported in Canada, one of the commonly grown varieties in Manitoba, "TH 32004", was selected for the pathogenicity test. Surface-sterilized soybean seeds, which had been pre-germinated for three days, were sown in a sterilized soilless growing mix (Sunshine #5) together with 5 g (approx. 1 × 105 microsclerotia) of macerated 10 to 14-day old inoculum grown on PDA-streptomycin agar medium at room temperature and applied using an inoculum layering technique. For the non-inoculated control, macerated PDA-streptomycin agar without mycelia was used. Twenty plants per treatment were maintained in a walk-in plant growth chamber with a 16 h photoperiod at 25/20 °C ± 1 °C (day/night) and 50% relative humidity. Plants were watered weekly but were subjected to water stress. Symptoms of charcoal rot were observed in the root systems of all inoculated soybean plants after 28 days, while no symptoms were observed in the control plants (Figure S1). There was production of microsclerotia on the roots inoculated with each isolate (data not shown). Three isolates of M. phaseolina were re-isolated from the inoculated plants and found to be identical to the inoculated isolates with respect to morphological characteristics in culture, as well as with respect to the ITS, TEF-1α and CAL DNA sequences. For each isolate and non-inoculated control, five seeds of 'TH 32004' were seeded per pot, and four pots were used for the inoculated and control treatments. The experiment was repeated twice in a randomized complete block design with similar results, fulfilling Koch's postulates. To our knowledge, this is the first report of charcoal rot caused by M. phaseolina on soybean in Manitoba, Canada.

First Report of Root Rot Caused by Macrophomina phaseolina in Peanut Shandong Province, China.

Peanut (Arachis hypogaea L.) is a widely grown oilseed crop of great agricultural importance worldwide. In July 2022, disease symptoms were observed on peanut roots in Laixi (36º85' N, 120º54' E), Shandong Province, China. About 25% of the plants showed various symptoms, including stem and root rot and blackening, microsclerotia on the stem, yellowing and wilting of leaves, and even death. Twenty diseased plants were collected to confirm the pathogen. Symptomatic roots were cut into small pieces, disinfested with 75% ethanol for 1 min and 0.5% NaClO for 2 min, rinsed three times with sterile water, dried on sterile filter paper, and then spread on potato dextrose agar (PDA) supplemented with 100 µg/mL chloramphenicol and incubated at 25°C in the dark. At the beginning of growth, the fungus formed sparse, white mycelia, which white, then darkened with age and microsclerotia were formed in the medium after 5 days. The mycelium aggregated into black, round to oblong or irregularly shaped microsclerotia 84 to 163 µm long and 54 to 125 µm wide (n=40). These morphological characteristics were consistent with the description of Macrophomina phaseolina (Holliday and Punithalingam, 1970). Molecular identification was performed by sequencing the internal transcribed spacer (ITS) region with ITS1 and ITS4 and translation elongation factor 1-alpha (TEF) with EF1-728F/EF1-986R (Glass and Donaldson 1995) of a representative isolate SXY183. ITS (OR056369) and TEF (OR098356) of SXY183 showed 100% and 97.74% similarity with M. phaseolina (KF951622, KF951997), respectively. Phylogenetic analysis was performed using Neighbor-Joining (NJ) analysis based on the gene sequences of ITS and TEF. The fungus was identified as M. phaseolina based on molecular analysis and morphological characteristics. The pathogenicity of a representative isolate (SXY183) was tested on peanuts under greenhouse conditions. Two-week-old peanut (Huayu No. 9115) seedlings were inoculated with a mycelial plug (8 mm diameter) at the root base of each plant and cultured in a greenhouse (30°C during the day and 25°C at night, a 12-h photoperiod, and 80% RH). Ten plants were inoculated with a plug of non-colonized PDA as a control. Brown lesions were observed on the stem and root of all inoculated seedlings 7 days after inoculation, but not on the control plants. The experiment was repeated three times. M. phaseolina was re-isolated from the symptomatic root and confirmed based on morphological characteristics and DNA sequence analysis of ITS and TEF. M. phaseolina is a soil-borne fungus that is distributed worldwide and has a broad host range. Disease agent has previously been reported on several host plants such as adzuki bean, faba bean, watermelon, Plukenetia volubilis, Atractylodes lancea and Curcuma longa in China (Cai et al., 2020; Sun et al. 2016; Sun et al., 2019; Sun et al., 2020; Wang et al., 2020; Wu et al., 2022). However, this is the first report in which M. phaseolina was found to cause peanut root rot in Shandong Province, China. Our report will provide important information for studying the epidemiology and management of this disease.

First Report of Charcoal Rot Caused by Macrophomina phaseolina on Stevia rebaudiana in Arizona, USA.

Stevia (Stevia rebaudiana Bertoni) is an important medicinal crop grown worldwide. Leaves of stevia contain a non-caloric sweetener, stevioside, which is used as a substitute to artificial sweeteners. In August 2022, symptoms of chlorosis, wilting, and root rot were observed in about 30 % of stevia plants growing at the Agricultural Station at Yuma Agricultural Center, Yuma, AZ, USA (32.7125° N, 114.7067° W). Infected plants initially showed chlorosis and wilting, and the plants eventually died with foliage remaining intact to the plant. Cross sections of the crown tissue of affected stevia plants showed necrotic tissue and a dark brown discoloration in areas of the vascular and cortical tissues. Dark brown microsclerotia were observed on stem bases and necrotic roots of the infected plants. Five symptomatic plants were sampled to isolate the pathogen. Root and crown tissues (0.5 to 1 cm) were surface disinfested with 1% sodium hypochlorite for 2 min, rinsed three times with sterile water, and plated onto potato dextrose agar (PDA). All the five isolates displayed rapid mycelial growth on PDA at 28°C with a 12-h photoperiod. The mycelia were initially hyaline and turned from gray to black after 7 days. Masses of dark spherical to oblong microsclerotia were observed after 3 days on PDA, measuring an average of 75 µm width × 114 µm length (n=30). For molecular identification, genomic DNA was extracted from mycelia and microsclerotia of a representative isolate (Yuma) using the DNeasy Plant Pro kit (Qiagen, Hilden, Germany). The internal transcribed spacer (ITS), translation elongation factor-1α (TEF-1α), calmodulin (CAL), and ß-tubulin (ß-TUB) regions were amplified using the primer sets, ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), MpCalF/MpCalR (Santos et al. 2020), and T1/T22 (O'Donnell and Cigelink 1997), respectively. A BLAST search of sequences revealed 98.7 to 100% identity to Macrophomina phaseolina sequences (MK757624, KT261797, MK447823, MK447918). Both morphological and molecular characteristics confirmed the fungus as M. phaseolina (Holliday and Punithaligam 1970). Sequences were submitted in the GenBank under accession numbers OP599770 (ITS), OP690156 (TEF-1α), OP612814 (CAL), and OP690157 (ß-TUB). Pathogenicity assay was performed on 9-week-old stevia plants (var. SW2267), grown in 4-inch planters in the greenhouse. The inoculum was made from a 14-day-old culture of M. phaseolina grown in conical flasks (250 ml) in potato dextrose broth at 28°C. Mycelial mats of the fungus were blended in 250 ml of sterile distilled water, filtered through four layers of cheesecloth, and then calibrated to 105 microsclerotia/ml using a hemocytometer. Twenty healthy plants were inoculated by soil drenching 50 ml of the inoculum per pot. Soil drenching using sterile distilled water was done on 5 non-inoculated control plants. Plants were maintained in the greenhouse at 28 ± 3°C with 12 h photoperiod. After 6 weeks, necrosis at the base of petioles and chlorosis of the leaves, followed by wilting were noticed on all 20 inoculated plants, whereas all the 5 control plants remained healthy. The fungus was reisolated and identified as M. phaseolina based on the morphology and sequences of ITS, TEF-1α, CAL and ß-TUB regions. Although M. phaseolina has been reported earlier on stevia in NC, USA (Koehler and Shew 2018), this is a first report from AZ, USA. M. phaseolina is known to be favored by high soil temperatures (Zveibil et al. 2011), thus represents a potential threat to stevia production in AZ, USA in coming years.

Charcoal Rot Severity and Soybean Yield Responses to Planting Date, Irrigation, and Genotypes.

Soybean (Glycine max [L.] Merr.) production is influenced by planting date, but its impact on yield in fields infested with Macrophomina phaseolina (Tassi) Goid. is unknown. A 3-year study was conducted in M. phaseolina-infested fields to assess the effects of planting date (PD) on disease severity and yield using eight genotypes, four of which are reported to be susceptible to charcoal rot (S), and four reported with moderate resistance (MR) to charcoal rot (CR). The genotypes were planted in early April, early May, and early June under irrigated and nonirrigated conditions. There was planting date by irrigation interaction for area under the disease progress curve (AUDPC) where May PD was significantly lower compared to April and June PDs in irrigated environments but not in nonirrigated environments. Correspondingly, yield in April PD was significantly lower than that of May and June. Interestingly, yield of S genotypes increased significantly with each subsequent PD, while yield of MR genotypes remained high across all three PDs. The interaction of genotypes by PD on yield revealed that the MR genotypes DT97-4290 and DS-880 had the greatest yields in May compared to April. While May PD had a decreased AUDPC and an increased yield across genotypes, the result of this research suggests that in fields infested with M. phaseolina, early May to early June planting coupled with appropriate cultivar selection provides maximum yield potential for western Tennessee and mid-southern soybean growers.