Transcriptional plasticity of schizotrophic Sclerotinia sclerotiorum responds to symptomatic rapeseed and endophytic wheat hosts.

IMPORTANCE: The broad host range of fungi with differential fungal responses leads to either a pathogenic or an endophytic lifestyle in various host plants. Yet, the molecular basis of schizotrophic fungal responses to different plant hosts remains unexplored. Here, we observed a general increase in the gene expression of S. sclerotiorum associated with pathogenicity in symptomatic rapeseed, including small protein secretion, appressorial formation, and oxalic acid toxin production. Conversely, in wheat, many carbohydrate metabolism and transport-associated genes were induced, indicating a general increase in processes associated with carbohydrate acquisition. Appressorium is required for S. sclerotiorum during colonization in symptomatic hosts but not in endophytic wheat. These findings provide new clues for understanding schizotrophic fungi, fungal evolution, and the emergence pathways of new plant diseases.

Zucchini Yellow Mosaic Virus (ZYMV) as a Serious Biotic Stress to Cucurbits: Prevalence, Diversity, and Its Implications for Crop Sustainability.

Zucchini yellow mosaic virus (ZYMV) is a severe threat to cucurbit crops worldwide, including Pakistan. This study was pursued to evaluate the prevalence, geographic distribution, and molecular diversity of ZYMV isolates infecting cucurbits in Pakistan's Pothwar region. Almost all the plant viruses act as a biotic stress on the host plants, which results in a yield loss. These viruses cause losses in single-infection or in mixed-infection cucurbit crops, and we have found a number of mixed-infected samples belonging to the Curubitaceae family. Serological detection of the tested potyviruses in the collected cucurbit samples revealed that ZYMV was the most prevalent virus, with a disease incidence (DI) at 35.2%, followed by Papaya ringspot virus (PRSV) with an incidence of 2.2%, and Watermelon mosaic virus (WMV) having an incidence as little as 0.5% in 2016. In the year 2017, a relatively higher disease incidence of 39.7%, 2.4%, and 0.3% for ZYMV, WMV, and PRSV, respectively, was recorded. ZYMV was the most prevalent virus with the highest incidence in Attock, Rawalpindi, and Islamabad, while PRSV was observed to be the highest in Islamabad and Jhelum. WMV infection was observed only in Rawalpindi and Chakwal. Newly detected Pakistani ZYMV isolates shared 95.8-97.0% nucleotide identities among themselves and 77.1-97.8% with other isolates retrieved from GenBank. Phylogenetic relationships obtained using different ZYMV isolates retrieved from GenBank and validated by in silico restriction analysis revealed that four Pakistani isolates clustered with other ZYMV isolates in group IIb with Chinese, Italian, Polish, and French isolates, while another isolate (MK848239) formed a separate minor clade within IIb. The isolate MK8482490, reported to infect bitter gourd in Pakistan, shared a minor clade with a Chinese isolate (KX884570). Recombination analysis revealed that the recently found ZYMV isolate (MK848239) is most likely a recombinant of Pakistani (MK848237) and Italian (MK956829) isolates, with a recombinant breakpoint between 266 and 814 nucleotide positions. Local isolate comparison and recombination detection may aid in the development of a breeding program that identifies resistant sources against recombinant isolates because the ZYMV is prevalent in a few cucurbit species grown in the surveyed areas and causes heavy losses and economic damage to the agricultural community.

Schizotrophic Sclerotinia sclerotiorum-Mediated Root and Rhizosphere Microbiome Alterations Activate Growth and Disease Resistance in Wheat.

Sclerotinia sclerotiorum, a widespread pathogen of dicotyledons, can grow endophytically in wheat, providing protection against Fusarium head blight and stripe rust and enhancing wheat yield. In this study, we found that wheat seed treatment with strain DT-8, infected with S. sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) and used as a "plant vaccine" for brassica protection, could significantly increase the diversity of the fungal and bacterial community in rhizosphere soil, while the diversity of the fungal community was obviously decreased in the wheat root. Interestingly, the relative abundance of potential plant growth-promoting rhizobacteria (PGPR) and biocontrol agents increased significantly in the DT-8-treated wheat rhizosphere soil. These data might be responsible for wheat growth promotion and disease resistance. These results may provide novel insights for understanding the interaction between the schizotrophic microorganism and the microbiota of plant roots and rhizosphere, screening and utilizing beneficial microorganisms, and further reducing chemical pesticide utilization and increasing crop productivity. IMPORTANCE Fungal pathogens are seriously threatening food security and natural ecosystems; efficient and environmentally friendly control methods are essential to increase world crop production. S. sclerotiorum, a widespread pathogen of dicotyledons, can grow endophytically in wheat, providing protection against Fusarium head blight and stripe rust and enhancing wheat yield. In this study, we discovered that S. sclerotiorum treatment increased the diversity of the soil fungal and bacterial community in rhizosphere soil, while the diversity of the fungal community was obviously decreased in the wheat root. More importantly, the relative abundance of potential PGPR and bio-control agents increased significantly in the S. sclerotiorum-treated wheat rhizosphere soil. The importance of this work is that schizotrophic S. sclerotiorum promotes wheat growth and enhances resistance against fungal diseases via changes in the structure of the root and rhizosphere microbiome.

Genetic diversity of cucumber green mottle mosaic virus (CGMMV) infecting cucurbits.

Cucumber green mottle mosaic virus (CGMMV), a well-known Tobamovirus, infects cucurbits across the globe. To determine its current status, molecular characterization, genetic recombination, gene flow and selection pressure, 10 districts from Punjab province of Pakistan were surveyed and a total of 2561 cucurbits samples were collected during 2019-2020. These samples were subjected to virus-specific double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) for the detection of CGMMV. The results revealed that viral disease was prevalent in all surveyed districts of Punjab with an overall 25.69% disease incidence. ELISA positive samples were further confirmed through RT-PCR and sequencing of coat protein (CP) cistron. Sequence analysis showed that the present studied CGMMV isolates have 96-99.5% nucleotide and 94.40-99.50% amino acid identities with those already available in GenBank. Phylogenetic analysis also revealed that understudied isolates were closely related with South Korean (AB369274) and Japanese (V01551) isolates and clustered in a separate clad. Sequence polymorphisms were observed in 663 bp of sequence within 31 CGMMV isolates covering complete CP gene. Total number of sites were 662, of which 610 and 52 sites were monomorphic and polymorphic (segregating), respectively. Of these polymorphic, 24 were singleton variable and 28 were parsimony informative. Overall nucleotide diversity (π) in all the understudied 31 isolates was 0.00010 while a total of 1 InDel event was observed and InDel Diversity (k) was 0.065. Haplotype diversity analysis revealed that there was a total 29 haplotypes with haplotype diversity (Hd) of 0.993458 in all the 31 isolates which provide evidence of less diversity among Pakistani isolates. The statistical analysis revealed the values 2.568, 5.31304 and 4.86698 of Tajima's D, Fu, & Li's F* and D*, respectively, which witnessed the population of CGMMV was under balanced selection pressure.

Enrichment of bacteria involved in the nitrogen cycle and plant growth promotion in soil by sclerotia of rice sheath blight fungus.

Rice sheath blight pathogen, Rhizoctonia solani, produces numerous sclerotia to overwinter. As a rich source of nutrients in the soil, sclerotia may lead to the change of soil microbiota. For this purpose, we amended the sclerotia of R. solani in soil and analyzed the changes in bacterial microbiota within the soil at different time points. At the phyla level, Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chloroflexi and Firmicutes showed varied abundance in the amended soil samples compared to those in the control. An increased abundance of ammonia-oxidizing bacterium (AOB) Nitrosospira and Nitrite oxidizing bacteria (NOB) i.e., Nitrospira was observed, where the latter is reportedly involved in the nitrifier denitrification. Moreover, Thiobacillus, Gemmatimonas, Anaeromyxobacter and Geobacter, the vital players in denitrification, N2O reduction and reductive nitrogen transformation, respectively, depicted enhanced abundance in R. solani sclerotia-amended samples. Furthermore, asymbiotic nitrogen-fixing bacteria, notably, Azotobacter as well as Microvirga and Phenylobacterium with nitrogen-fixing potential also enriched in the amended samples compared to the control. Plant growth promoting bacteria, such as Kribbella, Chitinophaga and Flavisolibacter also enriched in the sclerotia-amended soil. As per our knowledge, this study is of its kind where pathogenic fungal sclerotia activated microbes with a potential role in N transformation and provided clues about the ecological functions of R. solani sclerotia on the stimulation of bacterial genera involved in different processes of N-cycle within the soil in the absence of host plants.

Sclerotia of a phytopathogenic fungus restrict microbial diversity and improve soil health by suppressing other pathogens and enriching beneficial microorganisms.

Sclerotinia sclerotiorum, a notorious soil-borne pathogen of various important crops, produces numerous sclerotia to oversummer in the soil. Considering that sclerotia may also be attacked by other microbes in the soil, we hypothesized that sclerotia in soil may affect the community of soil microbes directly and/or indirectly. In this study, we inoculated sclerotia of S. sclerotiorum in soil collected from the field to observe changes in microbial diversity over three months using 16S rRNA and ITS2 sequencing techniques. Alpha diversity indices exhibited a decline in the diversity of microbial communities, while permanova results confirmed a significant difference in the microbial communities of sclerotia-amended and non-amended soil samples. In sclerotia-amended soil, fungal diversity showed enrichment of antagonists such as Clonostachys, Trichoderma, and Talaromyces and a drastic reduction in the plant pathogenic microbes compared to the non-amended soil. Sclerotia not only activated the antagonists but also enhanced the abundance of plant growth-promoting bacteria, such as Chitinophaga, Burkholderia, and Dyella. Moreover, the presence of sclerotia curtailed the growth of several notorious plant pathogenic fungi belonging to various genera such as Fusarium, Colletotrichum, Cladosporium, Athelia, Alternaria, and Macrophomina. Thus, we conclude that S. sclerotiorum when dormant in soil can reduce the diversity of soil microbes, including suppressing plant pathogens and enriching beneficial microbes. To the best of our knowledge, this is the first time a plant pathogen has been found in soil that can significantly suppress other pathogens. Our findings may provide novel cues to understand the ecology of crop pathogens in soil and maintaining soil conditions that could be beneficial for constructing a healthy soil microorganism community required for mitigating soil-borne diseases.

A concise review of biochar application to agricultural soils to improve soil conditions and fight pollution.

Application of biochar to soil can play a significant role in the alteration of nutrients dynamics, soil contaminants as well as microbial functions. Therefore, strategic biochar application to soil may provide agronomic, environmental and economic benefits. Key environmental outcomes may include reduced availability of toxic metals and organic pollutants, reduced soil N losses and longer-term storage of carbon in soil. The use of biochar can certainly address key soil agronomic constraints to crop production including Al toxicity, low soil pH and may improve nutrient use efficiency. Biochar application has also demerits to soil properties and attention should be paid when using a specific biochar for a specific soil property improvement. This review provides a concise assessment and addresses impacts of biochar on soil properties.

Antifungal Potential of Indigenous Medicinal Plants against Myrothecium Leaf Spot of Bitter Gourd ( Momordica charantia L.)

ABSTRACT Bitter gourd is of great importance due to its usage against the treatment of numerous ailments in human beings. A comprehensive survey at four localities of Southern Punjab, Pakistan was carried out to determine the severity of Myrothecium leaf spot. Maximum disease severity was at C1 (Chak 11/NP) and least at C2 (Kot Mehtab). Among isolated species Myrothecium roridum was found more prevalent and pathogenic as compared to M. verrucaria. Antifungal activity using solvent extracts of five medicinal plants (Mangifera indica, Melia azedarach, Nicotiana tabacum, Moringa oleifera and Eucalyptus globosum) were evaluated against isolated species by agar well diffusion method at various concentrations (0.01, 0.10, 1.0 and 10.0 µg / mL). N. tabacum revealed maximum zone size (13.40 mm and 8.28 mm) with ethanol and chloroform solvents respectively followed by M. azedarach (9.00mm and 6.48mm). However, least inhibition was observed with ethanol and chloroform extracts of E. globosum (6.04mm and 3.88mm zone size respectively). Ethanol extracts showed highest activity when compared to chloroform extracts. Qualitative phytochemical analysis showed that all the selected plants are rich in chemical compounds such as alkaloids, terpenoids, flavonoids and phenols whereas Saponins was only present in N. tabacum while absent in rest of the extracts.