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 %).

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.

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.

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.

Continuous microwave-assisted flow synthesis has been used as a simple, more efficient, and low-cost route to fabricate a range of nanosized (<100 nm) strontium-substituted calcium phosphates. In this study, fine nanopowder was synthesized via a continuous flow synthesis with microwave assistance from the solutions of calcium nitrate tetrahydrate (with strontium nitrate as Sr2+ ion source) and diammonium hydrogen phosphate at pH 10 with a time duration of 5 min. The morphological characterization of the obtained powder has been carried out by employing techniques such as transmission electron microscopy, X-ray diffraction, and Brunauer-Emmett-Teller surface area analysis. The chemical structural analysis to evaluate the surface properties was made by using X-ray photoelectron spectroscopy. Zeta potential analysis was performed to evaluate the colloidal stability of the particles. Antimicrobial studies were performed for all the compositions using four bacterial strains and an opportunistic human fungal pathogen Macrophomina phaseolina. It was found that the nanoproduct with high strontium content (15 wt% of strontium) showed pronounced antibacterial potential against M. luteus while it completely arrested the fungal growth after 48 h by all of its concentrations. Thus the synthesis strategy described herein facilitated the rapid production of nanosized Sr-substituted CaPs with excellent biological performance suitable for a bone replacement application.

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.

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.

3-Decalinoyltetramic acids (DTAs) are a class of natural products with chemical diversity and potent bioactivities. In fungal species there is a general biosynthetic route to synthesize this type of compounds, which usually features a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) and a lipocalin-like Diels-Alderase (LLDAse). Using a synthetic biology approach, combining the bioinformatics analysis prediction and heterologous expression, we mined a PKS-NRPS and LLDAse encoding gene cluster from the plant pathogenic fungus Macrophomina phaseolina and characterized the cluster to be responsible for the biosynthesis of novel DTAs, macrophasetins. In addition, we investigated the biosynthesis of these compounds and validated the accuracy of the phylogeny-guided bioinformatics analysis prediction. Our results provided a proof of concept example to this approach, which may facilitate the discovery of novel DTAs from the fungal kingdom.

Macrophomina phaseolina (M. phaseolina) is a crucial pathogenic fungus that can cause severe charcoal rot in economic crops and other plants. In this study, four new natural products, macrollins A-D, were discovered from M. phaseolina by the strategy of heterologous expression. To our knowledge, macrollins are the first reported polyketide-amino acid hybrids from the plant pathogen. Heterologous expression and in vitro reactions revealed a cytochrome P450 mono-oxygenase (MacC) catalyzing the hydroxylation at the β-carbon of tetramic acid molecules, which is different from P450s leading to the ring expansion in the biosynthesis of fungal 2-pyridones. Phylogenetic analysis of P450s involved in the fungal polyketide-amino acid hybrids showed that MacC was not classified in any known clades. The putative oxidative mechanisms of the P450s and the biosynthetic pathway of macrollins were also proposed.

In June 2018 and 2019, charcoal rot-like symptoms and black microsclerotia suggestive of Macrophomina phaseolina infection were observed on the basal stems of citrullus lanatus cv. \'Zaojia\', causing premature death. About 1 hectare of \'Zaojia\' had been investigated, disease incidence rates were almost 50%, resulting in a 40% yield loss in a single field in Shanghai, China (31°23\'N , 121°33\'E). A fungus was consistently isolated from infected watermelon tissues. In total, 30 cuttings from 10 infected seedlings were surface disinfected with 3% sodium hypochlorite for 3 min, washed thrice with sterile distilled water, air dried, and transferred onto potato dextrose agar (PDA). Dishes were incubated for 3 days at 27°C in the dark. Twenty four single hypha subcultures were obtained from these samples and were cultured for an additional 5 days at 27°C. Colonies were initially white, and then became grey black (Fig.1A). During the more advanced stages of infection, black microsclerotia were produced that were spherical or ovoid in shape (Fig.1B). No sexual structures and conidia developed during culture on PDA. Isolate pathogenicity was assessed both in vitro and in vivo. Watermelon plants (cv. \'Zaojia\') were grown in growth chambers at 28°C (day) and 23°C (night), with a 16 h photoperiod. When seedlings were 20 days old, they were inoculated. Briefly, a needle was used to puncture watermelon stems, and 5 mm agar plugs containing actively growing mycelia were placed on these needle wounds, followed by culture for 72 h at 27°C in a dark, humid chamber. In total, 10 seedlings were inoculated with 5 mm blank PDA, and the experiment was repeated three times, with the treatment being perfomed as described above. Seedling stems were inoculated 1-2 cm above the ground by puncturing them with a needle and then transferring 5 mm agar plugs containing fungi onto the wound sites. Seedlings were kept 75% humidity and then grown for 5 days at 27°C. Ten seedlings were inoculated per experiment. As a control, 10 seedlings were inoculated with 5 mm blank PDA plugs. Experiments were repeated three times. Necrotic spots around the wounds were evident on inoculated stems at 72 h (Fig.1C). Similarly, vascular tissue necrosis and the collapse of the surrounding pith and epidermis were observed on the residual parts of seedling stems after 5 days (Fig.1E), whereas control stems did not exhibit any disease related symptoms (Fig.1D, 1F). The same pathogen was then successfully re-isolated and was successfully regrown in pure culture, thus fulfilling Koch\'s postulates. To identify the causative pathogen, total mycelial DNA was isolated via the CTAB method (Brandfass & Karlovsky, 2008), and the internal transcribed spacer (ITS) rDNA regions were amplified using the ITS1/ITS4 primers (White, 1990), the genus-specific MPKFI//span>MpKRI primers were used for further amplification (Babu et al., 2007). M. phaseolina ITS sequences in this study shared 100% similarity with the ITS sequences of M. phaseolina from Chickpea (MK757624.1). Genus-specific sequences from this isolate shared 100% homology with other M. phaseolina isolates (MT645816.1 and MN263167.1). As such, M. phaseolina was confirmed to be the pathogen responsible for watermelon charcoal rot in the present report, which resulted in the death of infected watermelons before maturity, causing fruits to lose their commodity value. This report is the first to our knowledge to identify M. phaseolina as a causal pathogen of watermelon charcoal rot in China.