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.

The plant microbiome and plant-associated bacteria are known to support plant health, but there are limited studies on seed and seedling microbiome to reveal how seed-associated bacteria may confer disease resistance. In this study, the application of antibiotics on soybean seedlings indicated that seed-associated bacteria were involved in the seed rot resistance against a soil-borne pathogen Calonectria ilicicola, but this resistance cannot be carried to withstand root rot. Using PacBio 16S rRNA gene full-length sequencing and microbiome analyses, 14 amplicon sequence variants (ASVs) including 2 ASVs matching to Bacillus altitudinis were found to be more abundant in the 4 most resistant varieties versus the 4 most susceptible varieties. Culture-dependent isolation obtained two B. altitudinis isolates that both exhibit antagonistic capability against 6 fungal pathogens. Application of B. altitudinis on the most resistant and susceptible soybean varieties revealed different colonization compatibility, and the seed rot resistance was restored in the 5 varieties showing higher bacterial colonization. Moreover, qPCR confirmed the persistence of B. altitudinis on apical shoots till 21 days post-inoculation (dpi), but 9 dpi on roots of the resistant variety TN5. As for the susceptible variety HC, the persistence of B. altitudinis was only detected before 6 dpi on both shoots and roots. The short-term colonization of B. altitudinis on roots may explain the absence of root rot resistance. Collectively, this study advances the insight of B. altitudinis conferring soybean seed rot resistance and highlights the importance of considering bacterial compatibility with plant varieties and colonization persistence on plant tissues.

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 %).

RNA-Sequencing (RNA-Seq) and transcriptomic analyses have become powerful tools to study the developmental stages of fungal structures scuh as sclerotia. While RNA-Seq experiments have been set up for many important sclerotia- and microsclerotia-forming fungi, it has not been implemented to study Athelia rolfsii, which is one of the earliest fungi used in literature to uncover the roles of reactive oxygen species (ROS) in stimulating sclerotia formation. This study applied RNA-Seq to profile gene expression in four developmental stages of A. rolfsii sclerotia. Surprisingly, gene ontology and expression patterns suggested that most ROS-scavenging genes were not up-regulated in the stages from hyphal differentiation to the initial sclerotia stage. Using antioxidant and oxidant-amended culture assay, the results suggested none of the ascorbic acid, dithiothreitol (DTT), H2O2, or superoxide dismutase inhibitors [diethyldithiocarbamate (DETC), NaN3, and sodium dodecyl sulfate] affected the sclerotia number. Instead, only glutathione reduced the sclerotia number. Because glutathione has also been suggested to facilitate Ca2+ influx, therefore, glutathione culture assays with the combination of CaCl2, Ca2+-chelator egtazic acid, DETC, and H2O2 were tested on A. rolfsii, as well as two other fungi (Sclerotinia sclerotiorum and Macrophomina phaseolina) for comparison. Although the addition of CaCl2 caused sclerotia or microsclerotia reduction for all three fungi, the CaCl2-ROS interaction was only observed for S. sclerotiorum and M. phaseolina, but not A. rolfsi. Collectively, this study not only pointed out a conserved function of Ca2+ in suppressing fungal sclerotia and microsclerotia formation but also highlighted sclerotia formation of A. rolfsii being only sensitive to Ca2+ and independent of ROS stimuli.IMPORTANCEManagement for plant diseases caused by soil-borne fungal pathogens is challenging because many soil-borne fungal pathogens form sclerotia for long-term survival. Advanced understanding of the molecular and cellular mechanisms of sclerotia formation may provide novel insights to prevent these fungal residues in fields. This study discovered that Ca2+ acts as a negative signal cue to suppress sclerotia and microsclerotia formation in three economically important fungal pathogens. Moreover, the southern blight fungus Athelia rolfsii appears to be only regulated by Ca2+ but not reactive oxygen species. Accordingly, A. rolfsii can be a useful system for studying the detailed mechanism of Ca2+, and the applicability of Ca2+ in reducing sclerotia could be further assessed for disease management.

The combined stress studies provide fundamental knowledge that could assist in producing multiple stress resilient crops. The fungal phytopathogen, Macrophomina phaseolina is a major limiting factor in the productivity of the crop, Vigna radiata (mungbean). This fungal species tends to flourish under hot and dry conditions. Therefore, in this study the salicylic acid (SA) mediated stress responses in contrasting mungbean cultivars (Shikha and RMG-975) exposed to combined M. phaseolina infection (F) and drought stress (D) have been elucidated. The combined stress was applied to ten days seedlings in three orders i.e. drought followed by fungal infection (DF), drought followed by fungal infection with extended water deficit (DFD) and fungal infection followed by drought stress (FD). The severity of infection was analyzed using ImageJ analysis. Besides, the concentration of SA has been correlated with the phenylpropanoid pathway products, expression of pathogenesis-related proteins (β-1,3-glucanase and chitinase) and the specific activity of certain related enzymes (phenylalanine ammonia lyase, lipoxygenase and glutathione-S-transferase). The data revealed that the cultivar RMG-975 was relatively more tolerant than Shikha under individual stresses. However, the former became more susceptible to the infection under DFD treatment while the latter showed tolerance. Otherwise, the crown rot severity was reduced in both the cultivars under other combined treatments. The stress response analysis suggested that enhanced chitinase expression is vital for tolerance against both, the pathogen and drought stress. Also, it was noted that plants treat each stress combination differently and the role of SA was more prominently visible under individual stress conditions.

Charcoal rot disease (CRD), caused by the phytopathogenic fungus Macrophomina phaseolina, is a significant threat to cotton production in Israel and worldwide. The pathogen secretes toxins and degrading enzymes that disrupt the water and nutrient uptake, leading to death at the late stages of growth. While many control strategies were tested over the years to reduce CRD impact, reaching that goal remains a significant challenge. The current study aimed to establish, improve, and deepen our understanding of a new approach combining biological agents and chemical pesticides. Such intervention relies on reducing fungicides while providing stability and a head start to eco-friendly bio-protective Trichoderma species. The research design included sprouts in a growth room and commercial field plants receiving the same treatments. Under a controlled environment, comparing the bio-based coating treatments with their corresponding chemical coating partners resulted in similar outcomes in most measures. At 52 days, these practices gained up to 38% and 45% higher root and shoot weight and up to 78% decreased pathogen root infection (tracked by Real-Time PCR), compared to non-infected control plants. Yet, in the shoot weight assessment (day 29 post-sowing), the treatment with only biological seed coating outperformed (p < 0.05) all other biological-based treatments and all Azoxystrobin-based irrigation treatments. In contrast, adverse effects are observed in the chemical seed coating group, particularly in above ground plant parts, which are attributable to the addition of Azoxystrobin irrigation. In the field, the biological treatments had the same impact as the chemical intervention, increasing the cotton plants\' yield (up to 17%), improving the health (up to 27%) and reducing M. phaseolina DNA in the roots (up to 37%). When considering all treatments within each approach, a significant benefit to plant health was observed with the bio-chemo integrated management compared to using only chemical interventions. Specific integrated treatments have shown potential in reducing CRD symptoms, such as applying bio-coating and sprinkling Azoxystrobin during sowing. Aerial remote sensing based on high-resolution visible-channel (RGB), green-red vegetation index (GRVI), and thermal imaging supported the above findings and proved its value for studying CRD control management. This research validates the combined biological and chemical intervention potential to shield cotton crops from CRD.

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.

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).

Charcoal rot caused by Macrophomina phaseolina is one of the most devastating diseases that cause severe yield loss in Gloriosa superba cultivation. Plant growth-promoting rhizobacteria (PGPR) are extensively harnessed as biocontrol agents due to their effectiveness in combating a wide array of plant pathogens through a multifaceted approach. The present study delved into the mechanisms underlying its ability to inhibit root rot pathogen and its capacity to promote plant growth in G. superba, commonly known as glory lily. PGPR isolated from the rhizosphere of glory lily were subjected to in vitro assessments using the dual plate technique. The isolated Bacillus subtilis BGS-10 and B. velezensis BGS-21 showed higher mycelial inhibition (61%) against M. phaseolina. These strains also promote plant growth by producing indole-3-acetic acid, siderophore, ammonia, amylase, cellulase, pectinase, xylanase, and lipase chemicals. Genome screening of BGS-10 and BGS-21 revealed the presence of antimicrobial peptide genes such as Iturin (ituD gene), surfactin (srfA and sfp genes) along with the mycolytic enzyme β-1,3-glucanase. Further, the presence of secondary metabolites in the bacterial secretome was identified through gas chromatography-mass spectrometry (GC/MS) analysis. Notably, pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl), 9 H-pyrido[3,4-b] indole and L-leucyl-D-leucine exhibited the highest docking score against enzymes responsible for pathogen growth and plant cell wall degradation. Under glasshouse conditions, tuber treatment and soil application of talc-based formulation of B. subtilis BGS-10 and B. velezensis BGS-21 suppress the root rot incidence with a minimal disease incidence of 27.78% over untreated control. Concurrently, there was a notable induction of defense-related enzymes, including peroxidase (PO), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL), in glory lily. Therefore, it can be concluded that plant growth-promoting Bacillus strains play a significant role in fortifying the plant\'s defense mechanisms against the root rot pathogen.

Leucine-rich repeat receptor-like kinases (LRR-RLKs) can participate in the regulation of plant growth and development, immunity and signal transduction. Sesamum indicum, one of the most important oil crops, has a significant role in promoting human health. In this study, 175 SiLRR-RLK genes were identified in S. indicum, and they were subdivided into 12 subfamilies by phylogenetic analysis. Gene duplication analysis showed that the expansion of the SiLRR-RLK family members in the sesame was mainly due to segmental duplication. Moreover, the gene expansion of subfamilies IV and III contributed to the perception of stimuli under M. phaseolina stress in the sesame. The collinearity analysis with other plant species revealed that the duplication of SiLRR-RLK genes occurred after the differentiation of dicotyledons and monocotyledons. The expression profile analysis and functional annotation of SiLRR-RLK genes indicated that they play a vital role in biotic stress. Furthermore, the protein-protein interaction and coexpression networks suggested that SiLRR-RLKs contributed to sesame resistance to Macrophomina phaseolina by acting alone or as a polymer with other SiLRR-RLKs. In conclusion, the comprehensive analysis of the SiLRR-RLK gene family provided a framework for further functional studies on SiLRR-RLK genes.