Genomic surveillance uncovers a pandemic clonal lineage of the wheat blast fungus.

Wheat, one of the most important food crops, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America and to guide preemptive wheat breeding for blast resistance.

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.

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.

Development of a PCR-based assay for specific and sensitive detection of Fusarium buharicum from infected okra plant.

Fusarium wilt, caused by the fungus Fusarium buharicum, is an emerging disease of okra in Japan. The disease was first reported in Japan in 2015, causing significant damage to okra seedlings. Due to the potential threat in okra cultivation, the development of an accurate detection method for F. buharicum is needed for the surveillance and management of the disease. In this study, we designed a primer set and developed conventional and nested PCR assays for the specific detection of F. buharicum in infected okra plants and contaminated soil, respectively. We compared the diversity of the translation elongation factor 1 alpha (EF-1α) gene of F. buharicum with 103 other fungal species/isolates to design a species-specific primer. This primer pair successfully amplified approximately 400 bp of PCR product that was only detected in the F. buharicum isolate, not in the other fungal isolates. The developed nested PCR method was highly sensitive and could detect the fungus from a 0.01 fg DNA sample. The primer successfully detected the pathogen in artificially infected plants and soil by conventional and nested PCR, respectively. This is the first report of the development of the F. buharicum-specific primer set and detection assays, which can be used for the specific and sensitive detection of F. buharicum in field samples and for taking early control measures.

Enhancing rice growth and yield with weed endophytic bacteria Alcaligenes faecalis and Metabacillus indicus under reduced chemical fertilization.

Endophytic bacteria, recognized as eco-friendly biofertilizers, have demonstrated the potential to enhance crop growth and yield. While the plant growth-promoting effects of endophytic bacteria have been extensively studied, the impact of weed endophytes remains less explored. In this study, we aimed to isolate endophytic bacteria from native weeds and assess their plant growth-promoting abilities in rice under varying chemical fertilization. The evaluation encompassed measurements of mineral phosphate and potash solubilization, as well as indole-3-acetic acid (IAA) production activity by the selected isolates. Two promising strains, tentatively identified as Alcaligenes faecalis (BTCP01) from Eleusine indica (Goose grass) and Metabacillus indicus (BTDR03) from Cynodon dactylon (Bermuda grass) based on 16S rRNA gene phylogeny, exhibited noteworthy phosphate and potassium solubilization activity, respectively. BTCP01 demonstrated superior phosphate solubilizing activity, while BTDR03 exhibited the highest potassium (K) solubilizing activity. Both isolates synthesized IAA in the presence of L-tryptophan, with the detection of nifH and ipdC genes in their genomes. Application of isolates BTCP01 and BTDR03 through root dipping and spraying at the flowering stage significantly enhanced the agronomic performance of rice variety CV. BRRI dhan29. Notably, combining both strains with 50% of recommended N, P, and K fertilizer doses led to a substantial increase in rice grain yields compared to control plants receiving 100% of recommended doses. Taken together, our results indicate that weed endophytic bacterial strains BTCP01 and BTDR03 hold promise as biofertilizers, potentially reducing the dependency on chemical fertilizers by up to 50%, thereby fostering sustainable rice production.

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.

Morpho-molecular, cultural and pathological characterization of Athelia rolfsii causing southern blight disease on common bean.

Common bean (Phaseolus vulgaris), is a winter legume crop in Bangladesh and is considered an important vegetable with export potential. However, the production of common bean is severely affected by a newly reported soilborne fungal pathogen, Athelia rolfsii. This study aimed to characterize this new pathogen by morphological, molecular, cultural, and pathological analyses and determine the host range. The disease incidence in the affected field ranged between 6 and 13%. Initial disease symptoms were observed as brown sunken lesions at the point of infection and development of mycelia, followed by yellowing and quick wilting of the whole plant. A total of 10 fungal isolates were recovered from the infected plant samples, which were morphologically similar and produced white to brown mycelia and numerous brown sclerotia on the PDA medium. Two of them viz. BTCBSr3 and BTCBSr4 were used for the detailed study. Based on morphology and phylogenetic analyses of the sequenced data of internal transcribed spacer (ITS) and translation elongation factor 1 alpha (EF-1α), the pathogen was identified as A. rolfsii. Mycelial growth rate (3.6 cm/day) and fresh weight (107 mg) were higher in the PDA medium, whereas the number of sclerotia production (328/plate) was higher in OMA media. The isolates could grow in a wider range of incubation temperatures (15-35 °C) and media pH (3-9). In the cross-inoculation assay, both isolates were pathogenic on tomato, brinjal, and chickpea, but not on chili, soybean, and cowpea. This study has laid a foundation for further pathological research on the fungus in aid to develop an effective management practice against the pathogen.

Improvement of growth, yield and associated bacteriome of rice by the application of probiotic Paraburkholderia and Delftia.

Plant probiotic bacteria enhance growth and yield of crop plants when applied at the appropriate time and dose. Two rice probiotic bacteria, Paraburkholderia fungorum strain BRRh-4 and Delftia sp. strain BTL-M2 promote growth and yield of plants. However, no information is available on application of these two bacteria on growth, yield, and diversity and population of bacteriome in roots and rhizosphere soils of the treated rice plants. This study aimed to assess the effect of BRRh-4 and BTL-M2 application on growth, yield and bacteriome in roots and rhizosphere soil of rice under varying doses of N, P and K fertilizers. Application of BRRh-4 and BTL-M2 strains significantly (p < 0.05) increased seed germination, growth and yield of rice compared to an untreated control. Interestingly, the grain yield of rice by these bacteria with 50% less of the recommended doses of N, P, and K fertilizers were statistically similar to or better than the rice plants treated with 100% doses of these fertilizers. Targeted amplicon (16S rRNA) sequence-based analysis revealed significant differences (PERMANOVA, p = 0.00035) in alpha-diversity between the root (R) and rhizosphere soil (S) samples, showing higher diversity in the microbial ecosystem of root samples. Additionally, the bacteriome diversity in the root of rice plants that received both probiotic bacteria and chemical fertilizers were significantly higher (PERMANOVA, p = 0.0312) compared to the rice plants treated with fertilizers only. Out of 185 bacterial genera detected, Prevotella, an anaerobic and Gram-negative bacterium, was found to be the predominant genus in both rhizosphere soil and root metagenomes. However, the relative abundance of Prevotella remained two-fold higher in the rhizosphere soil metagenome (52.02%) than in the root metagenome (25.04%). The other predominant bacterial genera detected in the rice root metagenome were Bacillus (11.07%), Planctomyces (4.06%), Faecalibacterium (3.91%), Deinococcus (2.97%), Bacteroides (2.61%), and Chryseobacterium (2.30%). On the other hand, rhizosphere soil metagenome had Bacteroides (12.38%), Faecalibacterium (9.50%), Vibrio (5.94%), Roseomonas (3.40%), and Delftia (3.02%). Interestingly, we found the presence and/or abundance of specific genera of bacteria in rice associated with the application of a specific probiotic bacterium. Taken together, our results indicate that improvement of growth and yield of rice by P. fungorum strain BRRh-4 and Delftia sp. strain BTL-M2 is likely linked with modulation of diversity, structures, and signature of bacteriome in roots and rhizosphere soils. This study for the first time demonstrated that application of plant growth promoting bacteria significantly improve growth, yield and increase the diversity of bacterial community in rice.

In-silico prediction of highly promising natural fungicides against the destructive blast fungus Magnaporthe oryzae.

Magnaporthe oryzae causes destructive blast disease in more than 50 species of the major cereal crops rice, wheat and maize and destroys food of millions of people worldwide. Application of synthetic chemical fungicides are environmentally hazardous and unreliable in controlling M. oryzae. Conversely, naturally occurring biofungicides with multiple modes of actions are needed to be discovered for combatting the blast fungus. To find the effective biofungicides, we performed molecular docking study of some potential antifungal natural compounds targeting two proteins including a single-stranded DNA binding protein MoSub1 (4AGH), and an effector protein AVR-Pik (5E9G) of M. oryzae that regulates transcription in fungus and/or suppresses the host cell immunity. The thirty-nine natural compounds previously shown to inhibit M. oryzae growth and reproduction were put under molecular docking against these two proteins followed by simulation, free energy, and interaction analysis of protein-ligand complexes. The virtual screening revealed that two alkaloidal metabolites, camptothecin and GKK1032A2 showed excellent binding energy with any of these target proteins compared to reference commercial fungicides, azoxystrobin and strobilurin. Of the detected compounds, GKK1032A2 bound to both target proteins of M. oryzae. Both compounds showed excellent bioactivity scores as compared to the reference fungicides. Results of our computational biological study suggest that both camptothecin and GKK1032A2 are potential fungicides that could also be considered as lead compounds to design novel fungicides against the blast fungus. Furthermore, the GKK1032A2 acted as a multi-site mode of action fungicide against M. oryzae.

Zinc and Boron Soil Applications Affect Athelia rolfsii Stress Response in Sugar Beet (Beta vulgaris L.) Plants.

Generation of reactive oxygen species (ROS) constitutes an initial defense approach in plants during pathogen infection. Here, the effects of the two micronutrients, namely, zinc (Zn) and boron (B), on enzymatic and non-enzymatic antioxidant properties, as well as malondialdehyde (MDA) contents in leaves and roots challenged with Athelia rolfsii, which cause root rot disease, were investigated. The findings revealed that Zn and B application to the potting soil alleviated the adverse effect of A. rolfsii on sugar beet plants and increased the chlorophyll content in leaves. The increased enzymatic antioxidant activities such as catalase (CAT), peroxidase (POX), and ascorbate peroxidase (APX), and non-enzymatic antioxidants such as ascorbic acid (AsA) were observed in Zn applied plants compared to both uninoculated and inoculated control plants. A significant rise in CAT activity was noted in both leaves (335.1%) and roots (264.82%) due to the Zn2B1.5 + Ar treatment, in comparison to the inoculated control plants. On the other hand, B did not enhance the activity of any one of them except AsA. Meanwhile, A. rolfsii infection led to the increased accumulation of MDA content both in the leaves and roots of sugar beet plants. Interestingly, reduced MDA content was recorded in leaves and roots treated with both Zn and B. The results of this study demonstrate that both Zn and B played a vital role in A. rofsii tolerance in sugar beet, while Zn enhances antioxidant enzyme activities, B appeared to have a less pronounced effect on modulating the antioxidant system to alleviate the adverse effect of A. rolfsii.

Natural Protein Kinase Inhibitors, Staurosporine, and Chelerythrine Suppress Wheat Blast Disease Caused by Magnaporthe oryzae Triticum.

Protein kinases (PKs), being key regulatory enzymes of a wide range of signaling pathways, are potential targets for antifungal agents. Wheat blast disease, caused by Magnaporthe oryzae Triticum (MoT), is an existential threat to world food security. During the screening process of natural metabolites against MoT fungus, we find that two protein kinase inhibitors, staurosporine and chelerythrine chloride, remarkably inhibit MoT hyphal growth. This study further investigates the effects of staurosporine and chelerythrine chloride on MoT hyphal growth, conidia production, and development as well as wheat blast inhibition in comparison to a commercial fungicide, Nativo®75WG. The growth of MoT mycelia is significantly inhibited by these compounds in a dose-dependent manner. These natural compounds greatly reduce conidia production in MoT mycelia along with suppression of conidial germination and triggered lysis, resulting in deformed germ tubes and appressoria. These metabolites greatly suppress blast development in artificially inoculated wheat plants in the field. This is the first report of the antagonistic effect of these two natural PKC inhibitory alkaloids on MoT fungal developmental processes in vitro and suppression of wheat blast disease on both leaves and spikes in vivo. Further research is needed to identify their precise mechanism of action to consider them as biopesticides or lead compounds for controlling wheat blast.

Marine Natural Product Antimycin A Suppresses Wheat Blast Disease Caused by Magnaporthe oryzae Triticum.

The application of chemical pesticides to protect agricultural crops from pests and diseases is discouraged due to their harmful effects on humans and the environment. Therefore, alternative approaches for crop protection through microbial or microbe-originated pesticides have been gaining momentum. Wheat blast is a destructive fungal disease caused by the Magnaporthe oryzae Triticum (MoT) pathotype, which poses a serious threat to global food security. Screening of secondary metabolites against MoT revealed that antimycin A isolated from a marine Streptomyces sp. had a significant inhibitory effect on mycelial growth in vitro. This study aimed to investigate the inhibitory effects of antimycin A on some critical life stages of MoT and evaluate the efficacy of wheat blast disease control using this natural product. A bioassay indicated that antimycin A suppressed mycelial growth (62.90%), conidiogenesis (100%), germination of conidia (42%), and the formation of appressoria in the germinated conidia (100%) of MoT at a 10 µg/mL concentration. Antimycin A suppressed MoT in a dose-dependent manner with a minimum inhibitory concentration of 0.005 µg/disk. If germinated, antimycin A induced abnormal germ tubes (4.8%) and suppressed the formation of appressoria. Interestingly, the application of antimycin A significantly suppressed wheat blast disease in both the seedling (100%) and heading stages (76.33%) of wheat at a 10 µg/mL concentration, supporting the results from in vitro study. This is the first report on the inhibition of mycelial growth, conidiogenesis, conidia germination, and detrimental morphological alterations in germinated conidia, and the suppression of wheat blast disease caused by a Triticum pathotype of M. Oryzae by antimycin A. Further study is required to unravel the precise mode of action of this promising natural compound for considering it as a biopesticide to combat wheat blast.

Highly potent natural fungicides identified in silico against the cereal killer fungus Magnaporthe oryzae.

Magnaporthe oryzae is one of the most notorious fungal pathogens that causes blast disease in cereals, and results in enormous loss of grain production. Many chemical fungicides are being used to control the pathogen but none of them are fully effective in controlling blast disease. Therefore, there is a demand for the discovery of a new natural biofungicide to manage the blast disease efficiently. A large number of new natural products showed inhibitory activities against M. oryzae in vitro. To find out effective biofungicides, we performed in silico molecular docking analysis of some of the potent natural compounds targeting four enzymes namely, scytalone dehydratase, SDH1 (PDB ID:1STD), trihydroxynaphthalene reductase, 3HNR (PDB ID:1YBV), trehalose-6-phosphate synthase, Tps1 (PDB ID:6JBI) and isocitrate lyase, ICL1 (PDB ID:5E9G) of M. oryzae fungus that regulate melanin biosynthesis and/or appresorium formation. Thirty-nine natural compounds that were previously reported to inhibit the growth of M. oryzae were subjected to rigid and flexible molecular docking against aforementioned enzymes followed by molecular dynamic simulation. The results of virtual screening showed that out of 39, eight compounds showed good binding energy with any one of the target enzymes as compared to reference commercial fungicides, azoxystrobin and strobilurin. Among the compounds, camptothecin, GKK1032A2 and chaetoviridin-A bind with more than one target enzymes of M. oryzae. All of the compounds except tricyclazole showed good bioactivity score. Taken together, our results suggest that all of the eight compounds have the potential to develop new fungicides, and remarkably, camptothecin, GKK1032A2 and chaetoviridin-A could act as multi-site mode of action fungicides against the blast fungus M. oryzae.

Whole-genome sequencing of a year-round fruiting jackfruit (Artocarpus heterophyllus Lam.) reveals high levels of single nucleotide variation.

Jackfruit (Artocarpus heterophyllus Lam.) is the national fruit of Bangladesh and produces fruit in the summer season only. However, jackfruit is not commercially grown in Bangladesh because of an extremely high variation in fruit quality, short seasonal fruiting (June-August) and susceptibility to abiotic stresses. Conversely, a year-round high yielding (ca. 4-fold higher than the seasonal variety) jackfruit variety, BARI Kanthal-3 developed by the Bangladesh Agricultural Research Institute (BARI) derived from a wild accession found in Ramgarh of Chattogram Hiltracts of Bangladesh, provides fruits from September to June. This study aimed to generate a draft whole-genome sequence (WGS) of BARI Kanthal-3 to obtain molecular insights including genes associated with year-round fruiting trait of this important unique variety. The estimated genome size of BARI Kanthal-3 was 1.04-gigabase-pair (Gbp) with a heterozygosity rate of 1.62%. De novo assembly yielded a scaffolded 817.7 Mb genome while a reference-guided approach, yielded 843 Mb of genome sequence. The estimated GC content was 34.10%. Variant analysis revealed that BARI Kanthal-3 included 5.7 M (35%) and 10.4 M (65%) simple and heterozygous single nucleotide polymorphisms (SNPs), and about 90% of all these polymorphisms are in inter-genic regions. Through BUSCO assessment, 97.2% of the core genes were represented in the assembly with 1.3% and 1.5% either fragmented or missing, respectively. By comparing identified orthologous gene groups in BARI Kanthal-3 with five closely and one distantly related species of 10,092 common orthogroups were found across the genomes of the six species. The phylogenetic analysis of the shared orthogroups showed that A. heterophyllus was the closest species to BARI Kanthal-3 and orthogroups related to flowering time were found to be more highly prevalent in BARI Kanthal-3 compared to the other Arctocarpus spp. The findings of this study will help better understanding the evolution, domestication, phylogenetic relationships, year-round fruiting of this highly nutritious fruit crop as well as providing a resource for molecular breeding.

Bonactin and Feigrisolide C Inhibit Magnaporthe oryzae Triticum Fungus and Control Wheat Blast Disease.

Wheat blast caused by the Magnaporthe oryzaeTriticum (MoT) pathotype is one of the most damaging fungal diseases of wheat. During the screening of novel bioactive secondary metabolites, we observed two marine secondary metabolites, bonactin and feigrisolide C, extracted from the marine bacteria Streptomyces spp. (Act 8970 and ACT 7619), remarkably inhibited the hyphal growth of an MoT isolate BTJP 4 (5) in vitro. In a further study, we found that bonactin and feigrisolide C reduced the mycelial growth of this highly pathogenic isolate in a dose-dependent manner. Bonactin inhibited the mycelial development of BTJP 4 (5) more effectively than feigrisolide C, with minimal concentrations for inhibition being 0.005 and 0.025 µg/disk, respectively. In a potato dextrose agar (PDA) medium, these marine natural products greatly reduced conidia production in the mycelia. Further bioassays demonstrated that these secondary metabolites could inhibit the MoT conidia germination, triggered lysis, or conidia germinated with abnormally long branched germ tubes that formed atypical appressoria (low melanization) of BTJP 4 (5). Application of these natural products in a field experiment significantly protected wheat from blast disease and increased grain yield compared to the untreated control. As far as we are aware, this is the first report of bonactin and feigrisolide C that inhibited mycelial development, conidia production, conidial germination, and morphological modifications in the germinated conidia of an MoT isolate and suppressed wheat blast disease in vivo. To recommend these compounds as lead compounds or biopesticides for managing wheat blast, more research is needed with additional MoT isolates to identify their exact mode of action and efficacy of disease control in diverse field conditions.

Multistep regulation of protein kinase A in its localization, phosphorylation and binding with a regulatory subunit in fission yeast.

The cAMP-PKA is the major glucose-sensing pathway that controls sexual differentiation in Schizosaccharomyces pombe. Sequencing from the pka1 locus of recessive sam mutants, in which cells are highly inclined to sexual differentiation, led to the identification of mutations in the pka1 locus in sam5 (pka1-G441E) and sam7 (pka1-G441R). Rst2 and Ste11 proteins were induced and localized to the nucleus of sam5 and sam7 mutants even under rich glucose conditions, indicating that the function of Pka1 was completely abolished by mutations. Pka1-G441E and Pka1-G441R mutant proteins reside in the cytoplasm, even under glucose-rich conditions, while wild-type Pka1 resides in the nucleus, indicating that the functionality of Pka1 is important for its nuclear localization. This is supported by the observation that the Pka1-T356A mutant, which partially lacks Pka1 function, was localized to both the cytoplasm and the nucleus, but an active phosphomimetic Pka1-T356D mutant prtotein was localized to the nucleus under glucose-rich conditions. In addition to the basal phosphorylation of Pka1 at T356, hyperphosphorylation of Pka1 was observed under glucose-starved conditions, and such hyperphosphorylation was not observed in pka1-G441E, pka1-G441R, pka1-T356A or pka1-T356D mutants. As these mutant proteins failed to interact with a regulatory subunit Cgs1, hyperphosphorylation of Pka1 mutant proteins was considered to be dependent on Cgs1 interaction. Consistent with a role for Cgs1 in Pka1 phosphorylation, we detected the formation of a Cgs1-Pka1 complex prior to Pka1 hyperphosphorylation. Together, these results indicate that nuclear localization of Pka1 depends on its activity and hyperphosphorylation of Pka1 depends on Cgs1 interaction.

Complex formation, phosphorylation, and localization of protein kinase A of Schizosaccharomyces pombe upon glucose starvation.

Nine sam mutants that undergo sexual differentiation without requiring starvation in Schizosaccharomyces pombe were previously isolated. In this study, we identified a nonsense mutation on the pka1 locus in the sam6 mutant. pka1 encodes a catalytic subunit of protein kinase A (PKA). Replacement and overexpression of pka1 suppressed the KCl sensitivity and hyper-mating phenotype of sam6, confirming that sam6 is an allele of pka1. To characterize further the regulation of Pka1, we tested the physical interaction between Pka1 and Cgs1 (a regulatory subunit of PKA). Pka1 and Cgs1 physically interacted under glucose-limited conditions but not under glucose-rich conditions. In addition, the formation of a Pka1-Cgs1 complex was detected under glucose-limited conditions by Blue Native PAGE. Furthermore, the Pka1 protein was found to be phosphorylated under glucose-starved conditions, and at the same time its localization shifted from the nucleus towards the cytoplasm (mainly the vacuoles), suggesting a strong relationship among phosphorylation, complex formation, and the cytoplasmic distribution of Pka1.

Inhibitory Effects of Linear Lipopeptides From a Marine Bacillus subtilis on the Wheat Blast Fungus Magnaporthe oryzae Triticum.

Wheat blast is a devastating fungal disease caused by a filamentous fungus, Magnaporthe oryzae Triticum (MoT) pathotype, which poses a serious threat to food security of South America and South Asia. In the course of screening novel bioactive secondary metabolites, we found that some secondary metabolites from a marine Bacillus subtilis strain 109GGC020 remarkably inhibited the growth of M. oryzae Triticum in vitro at 20 µg/disk. We tested a number of natural compounds derived from microorganisms and plants and found that five recently discovered linear non-cytotoxic lipopeptides, gageopeptides A-D (1-4) and gageotetrin B (5) from the strain 109GGC020 inhibited the growth of MoT mycelia in a dose-dependent manner. Among the five compounds studied, gageotetrin B (5) displayed the highest mycelial growth inhibition of MoT followed by gageopeptide C (3), gageopeptide D (4), gageopeptide A (1), and gageopeptide B (2) with minimum inhibitory concentrations (MICs) of 1.5, 2.5, 2.5, 10.0, and 10.0 µg/disk, respectively. Application of these natural compounds has also completely blocked formation of conidia in the MoT fungal mycelia in the agar medium. Further bioassay revealed that these compounds (1-5) inhibited the germination of MoT conidia and, if germinated, induced deformation of germ tube and/or abnormal appressoria. Interestingly, application of these linear lipopeptides (1-5) significantly suppressed wheat blast disease on detached wheat leaves. This is the first report of the inhibition of mycelial growth, conidiogenesis, conidial germination, and morphological alterations in the germinated conidia and suppression of wheat blast disease by linear lipopeptides from the strain of B. subtilis. A further study is needed to evaluate the mode of action of these natural compounds for considering them as biopesticides for managing this notorious cereal killer.