Biological control potential of worrisome wheat blast disease by the seed endophytic bacilli.

Crop production often faces challenges from plant diseases, and biological control emerges as an effective, environmentally friendly, cost-effective, and sustainable alternative to chemical control. Wheat blast disease caused by fungal pathogen Magnaporthe oryzae Triticum (MoT), is a potential catastrophic threat to global food security. This study aimed to identify potential bacterial isolates from rice and wheat seeds with inhibitory effects against MoT. In dual culture and seedling assays, three bacterial isolates (BTS-3, BTS-4, and BTLK6A) demonstrated effective suppression of MoT growth and reduced wheat blast severity when artificially inoculated at the seedling stage. Genome phylogeny identified these isolates as Bacillus subtilis (BTS-3) and B. velezensis (BTS-4 and BTLK6A). Whole-genome analysis revealed the presence of genes responsible for controlling MoT through antimicrobial defense, antioxidant defense, cell wall degradation, and induced systemic resistance (ISR). Taken together, our results suggest that the suppression of wheat blast disease by seed endophytic B. subtilis (BTS-3) and B. velezensis (BTS-4 and BTLK6A) is liked with antibiosis and induced systemic resistance to wheat plants. A further field validation is needed before recommending these endophytic bacteria for biological control of wheat blast.

Suppressive Effects of Volatile Compounds from Bacillus spp. on Magnaporthe oryzae Triticum (MoT) Pathotype, Causal Agent of Wheat Blast.

The Magnaporthe oryzae Triticum (MoT) pathotype is the causal agent of wheat blast, which has caused significant economic losses and threatens wheat production in South America, Asia, and Africa. Three bacterial strains from rice and wheat seeds (B. subtilis BTS-3, B. velezensis BTS-4, and B. velezensis BTLK6A) were used to explore the antifungal effects of volatile organic compounds (VOCs) of Bacillus spp. as a potential biocontrol mechanism against MoT. All bacterial treatments significantly inhibited both the mycelial growth and sporulation of MoT in vitro. We found that this inhibition was caused by Bacillus VOCs in a dose-dependent manner. In addition, biocontrol assays using detached wheat leaves infected with MoT showed reduced leaf lesions and sporulation compared to the untreated control. VOCs from B. velezensis BTS-4 alone or a consortium (mixture of B. subtilis BTS-3, B. velezensis BTS-4, and B. velezensis BTLK6A) of treatments consistently suppressed MoT in vitro and in vivo. Compared to the untreated control, VOCs from BTS-4 and the Bacillus consortium reduced MoT lesions in vivo by 85% and 81.25%, respectively. A total of thirty-nine VOCs (from nine different VOC groups) from four Bacillus treatments were identified by gas chromatography-mass spectrometry (GC-MS), of which 11 were produced in all Bacillus treatments. Alcohols, fatty acids, ketones, aldehydes, and S-containing compounds were detected in all four bacterial treatments. In vitro assays using pure VOCs revealed that hexanoic acid, 2-methylbutanoic acid, and phenylethyl alcohol are potential VOCs emitted by Bacillus spp. that are suppressive for MoT. The minimum inhibitory concentrations for MoT sporulation were 250 mM for phenylethyl alcohol and 500 mM for 2-methylbutanoic acid and hexanoic acid. Therefore, our results indicate that VOCs from Bacillus spp. are effective compounds to suppress the growth and sporulation of MoT. Understanding the MoT sporulation reduction mechanisms exerted by Bacillus VOCs may provide novel options to manage the further spread of wheat blast by spores.

Role of seed infection for the near and far distance dissemination of wheat blast caused by Magnaporthe oryzae pathotype Triticum.

Magnaporthe oryzae pathotype Triticum (MoT) is a devastating fungal phytopathogen causing wheat blast disease which threatens wheat production particularly in warmer climate zones. Effective disease control is hampered by the limited knowledge on the life cycle, epidemiology, and pathogenicity of MoT. Since MoT mainly infects and colonizes the inflorescences of wheat, infection, invasion routes and colonization of MoT on wheat ears and in wheat seeds were investigated in order to assess potential seed transmission pathways. MoT was spray inoculated on two wheat cultivars (Sumai 3, susceptible and Milan, resistant) at three ear maturity stages [full ear emergence, growth stage (GS) 59; mid flowering, GS 65; and end of flowering, GS 69]. Incidence of MoT on Sumai 3 seeds was 100% and 20-25% on Milan. MoT sporulation rate on Sumai 3 contaminated seeds was more than 15 times higher than on Milan. Repeated washes of seed samples for removing paraffin fixation hampers seed microscopy. To overcome the damage of seed samples, we used hand-sectioned seed samples instead of paraffin-fixed microtome samples to facilitate microscopy. The colonization of MoT within various seed tissues was followed by light and confocal laser scanning microscopy (CLSM). Invasion of MoT in seeds predominantly occurred in the caryopsis germ region, but entry via other seed parts was also observed, confirming the potential of intense colonization of MoT in wheat grains. Fungal spread in wheat plants growing from MoT infected seeds was monitored through plating, microscopic and molecular techniques. Under greenhouse conditions, no spread of MoT from infected seeds to seedlings later than GS 21 or to ears was detected, neither in Milan nor in Sumai 3. We therefore conclude, that MoT may not systemically contaminate inflorescences and seeds in neither susceptible nor resistant wheat cultivars. However, initial blast symptoms, only found on seedlings of Sumai 3 but not Milan, resulted in the formation of new conidia, which may serve as inoculum source for plant-to-plant dissemination by airborne infection of plant stands in the field (short distance spread). Ultimately the inoculum may infect young inflorescences in the field and contaminate seeds. Our findings again stress the risk of long-distance dissemination of wheat blast across continents through MoT-contaminated seeds. This underlines the importance of mandatory use of healthy seeds in strategies to control any further spread of wheat blast.

Hydrogen peroxide detoxifying enzymes show different activity patterns in host and non-host plant interactions with Magnaporthe oryzae Triticum pathotype.

Wheat blast caused by the hemibiotroph fungal pathogen Magnaporthe oryzae Triticum (MoT) pathotype is a destructive disease of wheat in South America, Bangladesh and Zambia. This study aimed to determine and compare the activities of antioxidant enzymes in susceptible (wheat, maize, barley and swamp rice grass) and resistant (rice) plants when interacting with MoT. The activities of reactive oxygen species-detoxifying enzymes; catalase (CAT), ascorbate peroxidase (APX), glutathione peroxidase (GPX), glutathione S-transferase (GST), peroxidase (POX) were increased in all plants in response to MoT inoculation with a few exceptions. Interestingly, an early and very high activity of CAT was observed within 24 h after inoculation in wheat, barley, maize and swamp rice grass with lower H2O2 concentration. In contrast, an early and high accumulation of H2O2 was observed in rice at 48 hai with little CAT activity only at a later stage of MoT inoculation. The activities of APX, GST and POD were also high at an early stage of infection in rice. However, these enzymes activities were very high at a later stage in wheat, barley, maize and swamp rice grass. The activity of GPX gradually decreased with the increase of time in rice. Taken together, our results suggest that late and early inductions of most of the antioxidant enzyme activities occurs in susceptible and resistant plants, respectively. This study demonstrates some insights into physiological responses of host and non-host plants when interacting with the devastating wheat blast fungus MoT, which could be useful for developing blast resistant wheat.

First report of basal rot of dragon fruit caused by Fusarium oxysporum in Bangladesh.

Dragon fruit (Hylocereus polyrhizus) is a high value newly introduced fruit crop in Bangladesh. It has drawn considerable public attention due to its appealing flesh color, sweet taste and fruit qualities. Recently, basal rot of dragon fruit plants was observed in several farmer's fields, nurseries and in the research field of Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU) where about 10-15% of plants were infected in each location. Initially, the symptoms appeared in the basal part near the soil as brown lesions which gradually extended to the upper stem and finally becoming soft and watery (Figure 1a). Infected plants were collected from Kapasia of Gazipur district (Latitude 24.266 and Longitude 90.633) to isolate the causal organism. Isolations were carried out following the procedure reported by Briste et al. (2019). Briefly, infected plant parts were surface sterilized in 2% NaOCl for 1 min followed by 70% ethanol for 5 min and rinsed 3 times with sterile double distilled water. A large piece of a surface sterilized plant was cut into small pieces (2 mm × 2 mm) from the margin of the necrotic lesion and placed on half strength potato dextrose agar (PDA) and incubated for 7 days at 25 °C. The BTFD1 and BTFD4 isolates were purified from single spores resulting in white colonies with a growth rate of 1cm/day on PDA (Figure 1b). Colonies produced single celled microconidia from unbranched, short monophialidic conidiophores and septate macroconidia as well as chlamydospores in PDA which is consistent with Fusarium oxysporum (Figure 1c). To confirm the identity of the isolates, the internal transcribed spacer (ITS1, 5.8S rRNA and ITS2) and translation elongation factor-1alpha (EF-1α) were amplified using primers ITS-1/ ITS-4 and EF1-728F/ EF1-986R, respectively (Surovy et al. 2018). The ITS sequences of the isolates BTFD1 and BTFD4 (GenBank accession # MN727096 and MN727095, respectively) showed 100% similarity with the sequence from F. oxysporum strain JJF2 (MN626452). Sequence identity for EF-1α (GenBank accession # MN752123 and MN752124, respectively) was 100% with the sequence from F. oxysporum strain CAV041_EO (MK783088). The isolates (BTFD1 and BTFD4) were identified as F. oxysporum based on the aligned sequences of ITS and EF-1α, molecular phylogenetic analyses by maximum likelihood tree (Figure 2a) and maximum parsimony tree methods (Figure 2b). The isolates were stored at 4°C on dried filter paper as well as in an ultra-low temperature freezer (-80°C) at IBGE, BSMRAU, Bangladesh and are available on request. To ensure pathogenicity, isolate BTFD1 was grown on PDA, incubated at 25°C for 7 days and 250 ml conidial suspension (with 1 × 105 conidia/ml) was prepared. Twelve,three-month-old healthy dragon fruit plants were inoculated. Pathogenicity tests were carried out in two sets using three replications in each set. In one set, only the basal part of the plants was dipped into the conidial suspension and in another set the whole plant was dipped into the conidial suspension for two hours. Sterile distilled water was also used in another set of plants as a control. The inoculated plants were placed on wet tissue in a plastic box (31cm × 24cm × 8cm) covered and incubated at 25°C. After 10 days, all inoculated plants in both sets developed rot symptoms similar to those observed in the field, while the control plants remained healthy (Figure 1d). The pathogen was successfully re-isolated from the inoculated symptomatic parts on half strength PDA medium and had morphology as characterized before, thus fulfilling Koch's postulates. This disease has been reported in Argentina and Malaysia (Wright et al. 2007; Hafifi et al. 2019). To the bet of our knowledge, this is the first report of Fusarium basal rot of dragon fruit in Bangladesh caused by F. oxysporum.

Modulation of Nutritional and Biochemical Properties of Wheat Grains Infected by Blast Fungus Magnaporthe oryzae Triticum Pathotype.

Wheat blast disease caused by the Magnaporthe oryzae Triticum (MoT) pathotype exerts a significant impact on grain development, yield, and quality of the wheat. The aim of this study was to investigate morphological, physiological, biochemical, and nutritional properties of wheat cv. BARI Gom 24 under varying levels of blast disease severity in wheat spikes. Grain morphology, volume, weight, and germination of the infected grains were significantly affected by MoT. Biochemical traits specifically grain N, Ca, Mg, and Fe content significantly increased (up to threefold; p > 0.05), but organic carbon, Cu, Zn, B, and S content in wheat grains significantly decreased with increased severity of MoT infection. The grain crude protein content was about twofold higher (up to 18.5% in grain) in severely blast-infected grains compared to the uninfected wheat (9.7%). Analysis of other nutritional properties such as secondary metabolites revealed that total antioxidant activity, flavonoid, and carotenoid concentrations remarkably decreased (up to threefold) with increasing severity of blast infestation in wheat grain. Grain moisture, lipid, and ash content were slightly increased with the increase in blast severity. However, grain K and total phenolic concentration were increased at a certain level of blast infestation and then reduced with increase in MoT infestation.

Whole-Genome Sequence of a Plant Growth-Promoting Strain, Serratia marcescens BTL07, Isolated from the Rhizoplane of Capsicum annuum L.

Serratia marcescens strain BTL07, which has the ability to promote growth and suppress plant diseases, was isolated from the rhizoplane of a chili plant. The draft genome sequence data of the strain will contribute to advancing our understanding of the molecular mechanisms underlying plant growth promotion and tolerance to different stresses.

Cautionary Notes on Use of the MoT3 Diagnostic Assay for Magnaporthe oryzae Wheat and Rice Blast Isolates.

The blast fungus Magnaporthe oryzae is comprised of lineages that exhibit varying degrees of specificity on about 50 grass hosts, including rice, wheat, and barley. Reliable diagnostic tools are essential given that the pathogen has a propensity to jump to new hosts and spread to new geographic regions. Of particular concern is wheat blast, which has suddenly appeared in Bangladesh in 2016 before spreading to neighboring India. In these Asian countries, wheat blast strains are now co-occurring with the destructive rice blast pathogen raising the possibility of genetic exchange between these destructive pathogens. We assessed the recently described MoT3 diagnostic assay and found that it did not distinguish between wheat and rice blast isolates from Bangladesh. The assay is based on primers matching the WB12 sequence corresponding to a fragment of the M. oryzae MGG_02337 gene annotated as a short chain dehydrogenase. These primers could not reliably distinguish between wheat and rice blast isolates from Bangladesh based on DNA amplification experiments performed in separate laboratories in Bangladesh and in the United Kingdom. Specifically, all eight rice blast isolates tested in this study produced the WB12 amplicon. In addition, comparative genomics of the WB12 nucleotide sequence revealed a complex underlying genetic structure with related sequences across M. oryzae strains and in both rice and wheat blast isolates. We, therefore, caution against the indiscriminate use of this assay to identify wheat blast and encourage further development of the assay to ensure its value in diagnosis.

Emergence of wheat blast in Bangladesh was caused by a South American lineage of Magnaporthe oryzae.

BACKGROUND: In February 2016, a new fungal disease was spotted in wheat fields across eight districts in Bangladesh. The epidemic spread to an estimated 15,000 hectares, about 16 % of the cultivated wheat area in Bangladesh, with yield losses reaching up to 100 %. Within weeks of the onset of the epidemic, we performed transcriptome sequencing of symptomatic leaf samples collected directly from Bangladeshi fields. RESULTS: Reinoculation of seedlings with strains isolated from infected wheat grains showed wheat blast symptoms on leaves of wheat but not rice. Our phylogenomic and population genomic analyses revealed that the wheat blast outbreak in Bangladesh was most likely caused by a wheat-infecting South American lineage of the blast fungus Magnaporthe oryzae. CONCLUSION: Our findings suggest that genomic surveillance can be rapidly applied to monitor plant disease outbreaks and provide valuable information regarding the identity and origin of the infectious agent.

Macrocyclic Trichothecenes from Myrothecium roridum Strain M10 with Motility Inhibitory and Zoosporicidal Activities against Phytophthora nicotianae.

The cytotoxicity of the extract obtained from Myrothecium roridum M10 and a characteristic (1)H signal at δH ∼8 led to the assumption that verrucarin/roridin-type compounds were present. Upscaling on rice medium led to the isolation of four new metabolites: verrucarins Y (1) and Z (6) (macrocyclic trichothecenes), bilain D (12) (a diketopiperazine derivative), and hamavellone C (14) (an unusual cyclopropyl diketone). In addition, nine known trichothecenes [verrucarin A (3), 16-hydroxyverrucarin A (5), verrucarin B (7), 16-hydroxyverrucarin B (8), verrucarin J (2), verrucarin X (4), roridin A (9), roridin L-2 (10), and trichoverritone (11)] and a bicyclic lactone [myrotheciumone A (15)] were identified. Their structures and configurations were determined by spectroscopic methods, published data, Mosher's method, and considering biosyntheses. Some trichothecenes showed motility inhibition followed by lysis of the zoospores against devastating Phytophthora nicotianae within 5 min. Compounds 2, 3, 7, and 9 also exhibited potent activities against Candida albicans and Mucor miehei.

Gageopeptins A and B, new inhibitors of zoospore motility of the phytopathogen Phytophthora capsici from a marine-derived bacterium Bacillus sp. 109GGC020.

The motility of zoospores is critical in the disease cycles of the peronosporomycetes that cause devastating diseases in plants, fishes, vertebrates, and microbes. In the course of screening for secondary metabolites regulating the motility of zoospores of Phytophthora capsici, we discovered two new inhibitors from the ethyl acetate extract of the fermentation broth of a marine-derived strain Bacillus sp. 109GGC020. The structures of these novel metabolites were elucidated as new cyclic lipopeptides and named gageopeptins A (1) and B (2) by spectroscopic analyses including high resolution MS and extensive 1D and 2D NMR. The stereoconfigurations of 1 and 2 were assigned based on the chemical derivatization studies and reviews of the literature data. Although compounds 1 and 2 impaired the motility of zoospores of P. capsici in dose- and time-dependent manners, compound 1 (IC50 = 1 µg/ml) was an approximately 400-fold stronger motility inhibitor than 2 (IC50 = 400 µg/ml). Interestingly, the zoospores halted by compound 1 were subsequently lysed at higher concentrations (IC50 = 50 µg/ml). Compounds 1 and 2 were also tested against some bacteria and fungi by broth dilution assay, and exhibited moderate antibacterial and good antifungal activities.