Deciphering Molecular Determinants Underlying Penicillium digitatum’s Response to Biological and Chemical Antifungal Agents by Tandem Mass Tag (TMT)-Based High-Resolution LC-MS/MS

Penicillium digitatum is a widespread pathogen responsible for the postharvest decay of citrus, one of the most economically important crops worldwide. Currently, chemical fungicides are still the main strategy to control the green mould disease caused by the fungus. However, the increasing selection and proliferation of fungicide-resistant strains require more efforts to explore new alternatives acting via new or unexplored mechanisms for postharvest disease management. To date, several non-chemical compounds have been investigated for the control of fungal pathogens. In this scenario, understanding the molecular determinants underlying P. digitatum’s response to biological and chemical antifungals may help in the development of safer and more effective non-chemical control methods. In this work, a proteomic approach based on isobaric labelling and a nanoLC tandem mass spectrometry approach was used to investigate molecular changes associated with P. digitatum’s response to treatments with α-sarcin and beetin 27 (BE27), two proteins endowed with antifungal activity. The outcomes of treatments with these biological agents were then compared with those triggered by the commonly used chemical fungicide thiabendazole (TBZ). Our results showed that differentially expressed proteins mainly include cell wall-degrading enzymes, proteins involved in stress response, antioxidant and detoxification mechanisms and metabolic processes such as thiamine biosynthesis. Interestingly, specific modulations in response to protein toxins treatments were observed for a subset of proteins. Deciphering the inhibitory mechanisms of biofungicides and chemical compounds, together with understanding their effects on the fungal physiology, will provide a new direction for improving the efficacy of novel antifungal formulations and developing new control strategies.

Comparative genomics of the black rot pathogen Xanthomonas campestris pv. campestris and non-pathogenic co-inhabitant Xanthomonas melonis from Trinidad reveal unique pathogenicity determinants and secretion system profiles

Black-rot disease caused by the phytopathogen Xanthomonas campestris pv. campestris (Xcc) continues to have considerable impacts on the productivity of cruciferous crops in Trinidad and Tobago and the wider Caribbean region. While the widespread occurrence of resistance of Xcc against bactericidal agrochemicals can contribute to the high disease burdens, the role of virulence and pathogenicity features of local strains on disease prevalence and severity has not been investigated yet. In the present study, a comparative genomic analysis was performed on 6 pathogenic Xcc and 4 co-isolated non-pathogenic Xanthomonas melonis (Xmel) strains from diseased crucifer plants grown in fields with heavy chemical use in Trinidad. Native isolates were grouped into two known and four newly assigned ribosomal sequence types (rST). Mobile genetic elements were identified which belonged to the IS3, IS5 family, Tn3 transposon, resolvases, and tra T4SS gene clusters. Additionally, exogenous plasmid derived sequences with origins from other bacterial species were characterised. Although several instances of genomic rearrangements were observed, native Xcc and Xmel isolates shared a significant level of structural homology with reference genomes, Xcc ATCC 33913 and Xmel CFBP4644, respectively. Complete T1SS hlyDB, T2SS, T4SS vir and T5SS xadA, yapH and estA gene clusters were identified in both species. Only Xmel strains contained a complete T6SS but no T3SS. Both species contained a complex repertoire of extracellular cell wall degrading enzymes. Native Xcc strains contained 37 T3SS and effector genes but a variable and unique profile of 8 avr, 4 xop and 1 hpa genes. Interestingly, Xmel strains contained several T3SS effectors with low similarity to references including avrXccA1 (~89%), hrpG (~73%), hrpX (~90%) and xopAZ (~87%). Furthermore, only Xmel genomes contained a CRISPR-Cas I-F array, but no lipopolysaccharide wxc gene cluster. Xmel strains were confirmed to be non-pathogenic by pathogenicity assays. The results of this study will be useful to guide future research into virulence mechanisms, agrochemical resistance, pathogenomics and the potential role of the co-isolated non-pathogenic Xanthomonas strains on Xcc infections.

Apoplastic CBM1-interacting proteins bind conserved carbohydrate binding module 1 motifs in fungal hydrolases to counter pathogen invasion

When infecting plants, fungal pathogens secrete cell wall degrading enzymes (CWDEs) that break down cellulose and hemicellulose, the primary components of plant cell walls. Some fungal CWDEs contain a unique domain, named the carbohydrate binding module (CBM), that facilitates their access to polysaccharides. However, little is known about how plants counteract pathogen degradation of their cell walls. Here, we show that the rice cysteine-rich repeat secretion protein OsCBMIP binds to and inhibits xylanase MoCel10A of the blast fungus pathogen Magnaporthe oryzae, interfering with its access to the rice cell wall and degradation of rice xylan. We found binding of OsCBMIP to various CBM1-containing enzymes, suggesting it has a general role in inhibiting the catalytic activities of fungal enzymes. OsCBMIP is localized to the apoplast, and its expression is strongly induced in leaves infected with M. oryzae. Remarkably, knockdown of OsCBMIP reduced rice defense against M. oryzae, demonstrating that inhibition of CBM1-containing fungal enzymes by OsCBMIP is crucial for rice defense. We also identified additional CBMIP-related proteins from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism.

Nitrogen Fixing Bacteria and Their Application for Heavy Metal Removal: A Mini Review

Nitrogen is a critical component of biological systems and typically serves as a constraint on production in both aquatic and terrestrial environments, although its shortage has been compensated for through the process of biological nitrogen fixation. Nitrogen fixation is a critical microbial activity that utilises nitrogenase enzymes to convert dinitrogen (N2) gas to ammonia (NH3). It is carried out by a diverse spectrum of bacteria known as nitrogen fixing bacteria. These include free-living bacteria such as Azotobacter, Bacillus, Beijerickia, and Clostridium, associative bacteria such as Azospirillum, Enterobacter, and Pseudomonas, and bacteria that form symbiotic associations with legumes such as Rhizobium and actinorrhizal plants such as Frankia. These bacteria contribute significantly to plant growth by producing phytohormones (such as auxins, cytokinins, gibberelins, and indole acetic acid), reducing the incidence of plant diseases through the production of siderophores and cell wall degrading enzymes, and increasing phosphorus nutrition via phosphate solubilization. Additionally, they remove heavy metal ions from solutions through a process called biosorption, which is a feasible, natural, environmentally benign, and economically viable technique of remediating heavy metal-contaminated environments.

RNA-seq analysis of Rhizoctonia solani AG-4HGI strain BJ-1H infected by a new viral strain of Rhizoctonia solani partitivirus 2 reveals a potential mechanism for hypovirulence

Rhizoctonia solani partitivirus 2 (RsPV2), in the genus Alphapartitivirus, confers hypovirulence on Rhizoctonia solani AG-1-IA, the causal agent of rice sheath blight. In this study, a new strain of RsPV2 obtained from R. solani AG-4HGI strain BJ-1H, the causal agent of black scurf on potato, was identified and designated as Rhizoctonia solani partitivirus 2 strain BJ-1H (RsPV2-BJ). An RNA sequencing analysis of strain BJ-1H and the virus RsPV2-BJ-free strain BJ-1H-VF derived from strain BJ-1H was conducted to investigate the potential molecular mechanism of hypovirulence induced by RsPV2-BJ. In total, 14319 unigenes were obtained, and 1341 unigenes were identified as differentially expressed genes (DEGs), with 570 DEGs being down-regulated and 771 being up-regulated. Notably, several up-regulated DEGs were annotated to cell-wall-degrading enzymes, including β-1,3-glucanases. Strain BJ-1H exhibited increased expression of β-1,3-glucanase following RsPV2-BJ infection, suggesting that cell wall autolysis activity in R. solani AG-4HGI strain BJ-1H might be promoted by RsPV2-BJ, inducing hypovirulence in its host fungus R. solani AG-4HGI. To the best of our knowledge, this is the first report on the potential mechanism of hypovirulence induced by a mycovirus in R. solani.

Diversity and Plant Growth-Promoting Ability of Endophytic, Halotolerant Bacteria Associated with Tetragonia tetragonioides (Pall.) Kuntze

The diversity of salt-tolerant cultivable endophytic bacteria associated with the halophyte New Zealand spinach (Tetragonia tetragonioides (Pall.) Kuntze) was studied, and their plant beneficial properties were evaluated. The bacteria isolated from leaves and roots belonged to Agrobacterium, Stenotrophomonas, Bacillus, Brevibacterium, Pseudomonas, Streptomyces, Pseudarthrobacter, Raoultella, Curtobacterium, and Pantoea. Isolates exhibited plant growth-promoting traits, including the production of a phytohormone (indole 3-acetic-acid), cell wall degrading enzymes, and hydrogen cyanide production. Furthermore, antifungal activity against the plant pathogenic fungi Fusarium solani, F. oxysporum, and Verticillium dahliae was detected. Ten out of twenty bacterial isolates were able to synthesize ACC deaminase, which plays a vital role in decreasing ethylene levels in plants. Regardless of the origin of isolated bacteria, root or leaf tissue, they stimulated plant root and shoot growth under 200 mM NaCl conditions. Our study suggests that halophytes such as New Zealand spinach are a promising source for isolating halotolerant plant-beneficial bacteria, which can be considered as potentially efficient biofertilizers in the bioremediation of salt-affected soils.

Biocontrol Potential of Endophytic Actinobacteria against Fusarium solani, the Causal Agent of Sudden Decline Syndrome on Date Palm in the UAE

Thirty-one endophytic streptomycete and non-streptomycete actinobacteria were isolated from healthy date palm root tissues. In vitro screening revealed that the antifungal action of isolate #16 was associated with the production of cell-wall degrading enzymes, whereas with diffusible antifungal metabolites in isolate #28, albeit their production of volatile antifungal compounds. According to the 16S rRNA gene sequencing, isolates #16 and #28 were identified as Streptomyces polychromogenes UAE2 (Sp; GenBank Accession #: OK560620) and Streptomyces coeruleoprunus UAE1 (Sc; OK560621), respectively. The two antagonists were recovered from root tissues until 12 weeks after inoculation, efficiently colonized root cortex and xylem vessels, indicating that the date palm roots are a suitable habitat for these endophytic isolates. At the end of the greenhouse experiments, the development of sudden decline syndrome (SDS) was markedly suppressed by 53% with the application of Sp and 86% with Sc, confirming their potential in disease management. Results showed that the estimated disease severity indices in diseased seedlings were significantly (p < 0.05) reduced from 4.75 (scale of 5) to 2.25 or 0.67 by either Sp or Sc, respectively. In addition, conidial numbers of the pathogen significantly (p < 0.05) dropped by 38% and 76% with Sp and Sc, respectively, compared to infected seedlings with F. solani (control). Thus, the suppression of disease symptoms was superior in seedlings pre-inoculated with S. coeruleoprunus, indicating that the diffusible antifungal metabolites were responsible for F. solani retardation in these plants. This is the first report of actinobacteria naturally existing in date palm tissues acting as microbial antagonists against SDS on date palm.