Fluorescent Labeling of Peroxisome and Nuclear in Colletotrichum aenigma.

Anthracnose is one of the most widespread and destructive diseases in grapes. Grape anthracnose can be caused by various Colletotrichum species, such as Colletotrichum gloeosporioides and Colletotrichum cuspidosporium. In recent years, Colletotrichum aenigma was reported as a causal agent of Grape anthracnose in China and South Korea. Peroxisome is an important organelle in eukaryotes, which plays a very important role in the growth, development, and pathogenicity of several plant-pathogenic fungal species i, but it has not been reported in C. aenigma. In this work, the peroxisome of C. aenigma was labeled with a fluorescent protein, using green fluorescent protein (GFP) and red fluorescent protein (DsRED and mCherry) as reporter genes. Via Agrobacterium tumefaciens-mediated transformation (AtMT), two fluorescent fusion vectors to mark the peroxisomes, with GFP and DsRED, respectively, were introduced into a wild-type strain of C. aenigma. In the transformants, bright dots of green or red fluorescence in hyphae and spores could be seen in the strains labeled peroxisome. The nuclei labeled by the same method showed bright round fluorescent spots. In addition, we also combined fluorescent protein labeling with chemical staining to show the localization more clearly. The ideal peroxisome and nuclear fluorescence-labeled C. aenigma strain was obtained, which provided a reference for the study of its growth, development, and pathogenicity.

[Strategies for exogenous RNA delivery in RNAi-mediated pest management].

Plant diseases and insect pests threaten the safety of crop production greatly. Traditional methods for pest management are challenged by the problems such as environmental pollution, off-target effects, and resistance of pathogens and insects. New biotechnology-based strategies for pest control are expected to be developed. RNA interference (RNAi) is an endogenous process of gene regulation, which has been widely used to study the gene functions in various organisms. In recent years, RNAi-based pest management has received increasing attention. The effective delivery of the exogenous interference RNA into the targets is a key step in RNAi-mediated plant diseases and pest control. Considerable advances were made on the mechanism of RNAi, and various RNA delivery systems were developed for efficient pest control. Here we review the latest advances on mechanisms and influencing factors of RNA delivery, summarize the strategies of exogenous RNA delivery in RNAi-mediated pest control, and highlight the advantages of nanoparticle complexes in dsRNA delivery.

Advancements in the Use of Bacteriophages to Combat the Kiwifruit Canker Phytopathogen Pseudomonas syringae pv. actinidiae.

Over the last several decades, kiwifruit production has been severely damaged by the bacterial plant pathogen Pseudomonas syringae pv. actinidiae (Psa), resulting in severe economic losses worldwide. Currently, copper bactericides and antibiotics are the main tools used to control this bacterial disease. However, their use is becoming increasingly ineffective due to the emergence of antibiotic resistance. In addition, environmental issues and the changes in the composition of soil bacterial communities are also concerning when using these substances. Although biocontrol methods have shown promising antibacterial effects on Psa infection under in vitro conditions, the efficiency of antagonistic bacteria and fungi when deployed under field conditions remains unclear. Therefore, it is crucial to develop a phage-based biocontrol strategy for this bacterial pathogen. Due to the specificity of the target bacteria and for the benefit of the environment, bacteriophages (phages) have been widely regarded as promising biological agents to control plant, animal, and human bacterial diseases. An increasing number of studies focus on the use of phages for the control of plant diseases, including the kiwifruit bacterial canker. In this review, we first introduce the characteristics of the Psa-induced kiwifruit canker, followed by a description of the diversity and virulence of Psa strains. The main focus of the review is the description of recent advances in the isolation of Psa phages and their characterization, including morphology, host range, lytic activity, genome characterization, and lysis mechanism, but we also describe the biocontrol strategies together with potential challenges introduced by abiotic factors, such as high temperature, extreme pH, and UV irradiation in kiwifruit orchards. The information presented in this review highlights the potential role of phages in controlling Psa infection to ensure plant protection.

Monitoring peroxisome dynamics using enhanced green fluorescent protein labeling in Alternaria alternata.

Brown leaf spot on tobacco is a serious fungal disease caused by Alternaria alternata. Peroxisomes are organelles playing an important role in the development and infection of plant pathogenic fungi. But, until now, there is no report on the peroxisome dynamics during the conidia germination of A. alternata. To evaluate the roles of peroxisome in the development of the fungus, in the present work, an enhanced green fluorescent protein (eGFP) cassette tagged with peroxisome targeting signal 2 (PTS2) was integrated into A. alternata to label the organelles, and an eGFP cassette carrying a nuclear located signal (NLS) was performed parallelly. The transformants containing the fusions emitted fluorescence in punctate patterns. The fluorescence of eGFP-PTS2 was distributed exactly in the peroxisomes while those of eGFP-NLS were located in the nucleus. Typical AaGB transformants were selected to be investigated for the peroxisome dynamics. The results showed that during spore germination, the number of peroxisomes in the spores decreased gradually, but increased in the germ tubes. In addition, when the transformants were cultured on lipid media, the numbers of peroxisomes increased significantly, and in a larger portion, present in striped shapes. These findings give some clues for understanding the peroxisomal functions in the development of A. alternata.

Inhibitory Effect and Mechanism of Trichoderma taxi and Its Metabolite on Trichophyton mentagrophyte.

Trichophyton mentagrophytes is an important zoonotic dermatophyte, which seriously harms the skin of humans and animals. Chemical drugs are generally used for the prevention and treatment of the disease caused by T. mentagrophytes. Discovering new compounds from natural products is an important approach for new drug development. Trichoderma includes a variety of fungal species used for biological control of phytopathogenic fungi. However, the antifungal effects of Trichoderma and their metabolites on zoonotic fungal pathogens are largely unknown. Here, the effect of trichodermin, a metabolite derived from the plant endophytic fungus Trichoderma taxi, on T. mentagrophytes was examined, and the underlying mechanism was explored. T. mentagrophytes growth was suppressed significantly by trichodermin and completely inhibited under 1000 µg/mL trichodermin. The production and germination of T. mentagrophytes spores were remarkably reduced upon exposure to trichodermin, in comparison with control samples. Treatment of lesions caused by T. mentagrophytes on the rabbit skin with 1 mg/mL trichodermin prompted the healing process significantly; however, 20 mg/mL trichodermin was likely toxic to the skin. Under trichodermin treatment, the number of mitochondria in T. mentagrophytes increased significantly, while a few mitochondria-related genes decreased, indicating possible mitochondrial damage. In transcriptome analysis, the GO terms enriched by DEGs in the trichodermin-treated group included carbohydrate metabolic process, integral component of membrane, intrinsic component of membrane, and carbohydrate binding, while the enriched KEGG pathways comprised biosynthesis of secondary metabolites, glycolysis/gluconeogenesis, and carbon metabolism. By comparing the wild type and a gene deletion strain of T. mentagrophytes, we found that CDR1, an ABC transporter encoding gene, was involved in T. mentagrophytes sensitivity to trichodermin.

The peroxins BcPex8, BcPex10, and BcPex12 are required for the development and pathogenicity of Botrytis cinerea.

Peroxisomes have been proved playing roles in infection of several plant pathogens. Although the contribution of a portion of peroxins in pathogenicity was demonstrated, most of them are undocumented in fungi, especially, Botrytis cinerea. The homologs of Pex8, Pex10, and Pex12 in B. cinerea were functionally characterized in this work using gene disruption strategies. Compared with the wild-type strain (WT), the Δbcpex8, Δbcpex10, and Δbcpex12 mutants exhibited significant reduction in melanin production, fatty acid utilization, and decreased tolerance to high osmotic pressure and reactive oxygen species (ROS). The mycelial growth and conidiation of were significantly inhibited in Δbcpex8, Δbcpex10, and Δbcpex12 strains. The mycelial growth rates of Δbcpex8, Δbcpex10, and Δbcpex12 were reduced by 32, 35, and 34%, respectively, compared with WT and ectopic transformant (ET), and the conidiation was reduced by approximately 89, 27, and 88%, respectively. The conidial germination, germ tube elongation, and the formation of initiate infection structures (IFSs) were also reduced by the deletion of the genes. The pathogenicity was tested on the leaves of tobacco and strawberry, and fruits of tomato. On the leaves of tobacco and strawberry, the Δbcpex8, Δbcpex10, and Δbcpex12 mutants could not induce necrotic lesions, and the lesions on tomato fruits infected with the mutants were significantly reduced than those of the wide type. The results indicated that BcPEX8, BcPEX10, and BcPEX12 are indispensable for the development and pathogenicity of B. cinerea.

MoDHX35, a DEAH-Box Protein, Is Required for Appressoria Formation and Full Virulence of the Rice Blast Fungus, Magnaporthe oryzae.

The DExD/H-box protein family encompasses a large number of RNA helicases that are involved in RNA metabolism and a variety of physiological functions in different species. However, there is limited knowledge of whether DExD/H-box proteins play a role in the pathogenicity of plant fungal pathogens. In the present work, the DExD/H-box protein MoDHX35, which belongs to the DEAH subfamily, was shown to be crucial in appressoria formation and full virulence of the rice blast fungus, Magnaporthe oryzae. The predicted protein sequence of MoDHX35 had typical DEAH-box domains, showed 47% identity to DHX35 in Homo species, but had no orthologs in Saccharomyces cerevisiae. Deletion of the MoDHX35 gene resulted in reduced tolerance of the mutants to doxorubicin, a nucleic acid synthesis disturbing agent, suggesting the involvement of MoDHX35 in RNA metabolism. MoDHX35-deleted mutants exhibited normal vegetative growth, conidia generation and conidial germination, but showed a reduced appressorium formation rate and attenuated virulence. Our work demonstrates the involvement of DEAH-box protein functions in the pathogenicity of plant fungal pathogens.

Adenylsuccinate Synthetase MoADE12 Plays Important Roles in the Development and Pathogenicity of the Rice Blast Fungus.

Purines are basic components of nucleotides in living organisms. In this study, we identified the ortholog of adenylosuccinate synthase MoADE12 in Magnaporthe oryzae by screening for growth-defective T-DNA insertional mutants. Gene replacement was performed to investigate the biological role of MoADE12. Δmoade12 mutants were adenine auxotrophs that failed to produce conidia, and showed reduced perithecia formation and pathogenicity. Moreover, the Δmoade12 mutant was hypersensitive to Congo red and oxidants, indicating that MoADE12 was required for cell wall integrity and oxidative stress resistance. Transcriptomic analysis identified the underlying mechanisms and indicated that several pathogenicity-related genes were regulated in the Δmoade12 mutant. Therefore, our data suggest that the adenylosuccinate synthase MoADE12 is involved in the de novo AMP biosynthesis pathway and is important for conidiation and pathogenicity in the rice blast fungus.

Development of Droplet Digital PCR Assay for Detection of Seed-Borne Burkholderia glumae and B. gladioli Causing Bacterial Panicle Blight Disease of Rice.

Bacterial panicle blight of rice or bacterial grain rot of rice is a worldwide rice disease. Burkholderia glumae and B. gladioli are the causal agents. The early and accurate detection of seed-borne B. glumae and B. gladioli is critical for domestic and international quarantine and effective control of the disease. Here, genomic analyses revealed that B. gladioli contains five phylogroups and the BG1 primer pair designed to target the 3'-end sequence of a gene encoding a Rhs family protein is specific to B. glumae and two phylogroups within B. gladioli. Using the BG1 primer pair, a 138-bp DNA fragment was amplified only from the tested panicle blight pathogens B. glumae and B. gladioli. An EvaGreen droplet digital PCR (dPCR) assay on detection and quantification of the two pathogens was developed from a SYBR Green real-time quantitative PCR (qPCR). The detection limits of the EvaGreen droplet dPCR on the two pathogens were identical at 2 × 103 colony forming units (CFU)∙mL-1 from bacterial suspensions and 2 × 102 CFU∙seed-1 from rice seeds. The EvaGreen droplet dPCR assay showed 10-fold detection sensitivity of the SYBR Green qPCR and could detect a single copy of the target gene in a 20-µL assay. Together, the SYBR Green qPCR assay allows for routine high-throughput detection of the panicle blight pathogens and the EvaGreen droplet dPCR assay provides a high-sensitive and high-accurate diagnostic method for quarantine of the pathogens.

The flavohemoglobin gene MoFHB1 is involved in the endurance against nitrosative stress in Magnaporthe oryzae.

Nitric oxide (NO) homeostasis plays a versatile role in pathogen-host interactions. To maintain NO homeostasis in favor of pathogens, microbes have evolved NO degradation systems besides NO synthesis pathway, in which the flavohemoglobin and S-nitrosoglutathione (GSNO) reductase are two key enzymes. We previously proved that MoSFA1, a GSNO reductase, is required for the growth and pathogenicity in Magnaporthe oryzae. In the present work, MoFHB1, a flavohemoglobin-encoding gene in M. oryzae was functionally characterized. Although the expression of the MoFHB1 gene was developmentally regulated during conidial germination and appressorium development, disruption of MoFHB1 did not change vegetative growth, conidiation and virulence. However, compared with the Δmosfa1 mutant, the Δmofhb1 mutant was significantly more sensitive to NO stress, and the expression of MoSFA1 gene in the Δmofhb1 mutant was significantly upregulated. Double deletion of MoSFA1 and MoFHB1 led to greater sensitivity of the fungus to NO stress than either of the single gene mutant, but no further reduction in pathogenicity was found compared with that of Δmosfa1 mutant. Taken together, MoFHB1 played an important role in NO detoxification but was dispensable for virulence of M. oryzae.

Identification and Characterization of a New Type of Holin-Endolysin Lysis Cassette in Acidovorax oryzae Phage AP1.

Phages utilize lysis systems to allow the release of newly assembled viral particles that kill the bacterial host. This is also the case for phage AP1, which infects the rice pathogen Acidovorax oryzae. However, how lysis occurs on a molecular level is currently unknown. We performed in silico bioinformatics analyses, which indicated that the lysis cassette contains a holin (HolAP) and endolysin (LysAP), which are encoded by two adjacent genes. Recombinant expression of LysAP caused Escherichia coli lysis, while HolAP arrested growth. Co-expression of both proteins resulted in enhanced lysis activity compared to the individual proteins alone. Interestingly, LysAP contains a C-terminal region transmembrane domain, which is different from most known endolysins where a N-terminal hydrophobic region is found, with the potential to insert into the membrane. We show that the C-terminal transmembrane domain is crucial for protein localization and bacterial lysis in phage AP1. Our study characterizes the new phage lysis cassette and the mechanism to induce cell disruption, giving new insight in the understanding of phage life cycles.

Current trends and future prospective in nanoremediation of heavy metals contaminated soils: A way forward towards sustainable agriculture.

Heavy metals (HMs) contamination in agricultural soils is a major concern for global food safety and human health. Although, various in-situ and ex-situ remediation methods have been used for the treatment of HMs contaminated soils, however, they also have many drawbacks viz., capital investment, toxicity, and environmental health hazards. Consequently, there is an urgent need to develop a novel method to ameliorate the toxicity of HMs in agricultural soils. In recent years, nanoparticles (NPs) have gained significant attention due to their potential applications in the environment and agriculture fields. Nanoremediation employs NPs that effectively reduce the contents of toxic HMs in the soil-plant system. Several studies have reported that the application of NPs in HMs-polluted soils, which reduced plant-available HMs concentration soils. However, the long-term efficiency of NPs immobilization is still unclear. Here, we provide details about the toxicity of HMs to environmental systems and potential applications NPs to alleviate the accumulation of HMs in agricultural soils. Finally, we present the mechanistic route of HMs-toxicity alleviation in plants by NPs application as well as their long-term efficiency and future prospects.

MAT Loci Play Crucial Roles in Sexual Development but Are Dispensable for Asexual Reproduction and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae.

Magnaporthe oryzae, a fungal pathogen that causes rice blast, which is the most destructive disease of rice worldwide, has the potential to perform both asexual and sexual reproduction. MAT loci, consisting of MAT genes, were deemed to determine the mating types of M. oryzae strains. However, investigation was rarely performed on the development and molecular mechanisms of the sexual reproduction of the fungus. In the present work, we analyzed the roles of two MAT loci and five individual MAT genes in the sex determination, sexual development and pathogenicity of M. oryzae. Both of the MAT1-1 and MAT1-2 loci are required for sex determination and the development of sexual structures. MAT1-1-1, MAT1-1-3 and MAT1-2-1 genes are crucial for the formation of perithecium. MAT1-1-2 impacts the generation of asci and ascospores, while MAT1-2-2 is dispensable for sexual development. A GFP fusion experiment indicated that the protein of MAT1-1-3 is distributed in the nucleus. However, all of the MAT loci or MAT genes are dispensable for vegetative growth, asexual reproduction, pathogenicity and pathogenicity-related developments of the fungus, suggesting that sexual reproduction is regulated relatively independently in the development of the fungus. The data and methods of this work may be helpful to further understand the life cycle and the variation of the fungus.

Effect of the Nanoparticle Exposures on the Tomato Bacterial Wilt Disease Control by Modulating the Rhizosphere Bacterial Community.

Ralstonia Solanacearum is one of the most infectious soil-borne bacterial plant pathogens, causing tomato bacterial wilt (TBW). Nanotechnology is an emerging area of research, particularly the application of nanoparticles (NPs) as nanopesticides to manage plant disease is gaining attention nowadays. However, the interaction between NPs and rhizosphere bacterial communities remains largely elusive. This study indicated that metal NPs (CuO, ZnO, and FeO) reduced the incidence of bacterial wilt to varying degrees and affected the composition and structure of the rhizosphere bacterial community. The results revealed that the application of metal oxide NPs can improve the morphological and physiological parameters of TBW infected tomato plants. Among all, CuONPs amendments significantly increase the Chao1 and Shannon index. In the early stage (the second week), it significantly reduces the relative abundance of pathogens. However, the relative abundance of beneficial Streptomyces bacteria increased significantly, negatively correlated with the relative abundance of pathogenic bacteria. In addition, the nano-treatment group will enrich some potential beneficial bacteria such as species from Sphingomonadaceae, Rhizobiaceae, etc. In general, our research provides evidence and strategies for preventing and controlling soil-borne disease tomato bacterial wilt with metal oxide NPs.

Identification of Genes Involved in Antifungal Activity of Burkholderia seminalis Against Rhizoctonia solani Using Tn5 Transposon Mutation Method.

Rhizoctonia solani is the causative agent of rice sheath blight disease. In a previous study, we found that the growth of R. solani was inhibited by Burkholderia seminalis strain R456. Therefore, the present study was conducted to identify the genes involved in the antifungal activity of B. seminalis strain R456 by using a Tn5 transposon mutation method. Firstly, we constructed a random insertion transposon library of 997 mutants, out of which 11 mutants showed the defective antifungal activity against R. solani. Furthermore, the 10 antagonism-related genes were successfully identified based on analysis of the Tn5 transposon insertion site. Indeed, this result indicated that three mutants were inserted on an indigenous plasmid in which the same insertion site was observed in two mutants. In addition, the remaining eight mutants were inserted on different genes encoding glycosyl transferase, histone H1, nonribosomal peptide synthetase, methyltransferase, MnmG, sulfate export transporter, catalase/peroxidase HPI and CysD, respectively. Compared to the wild type, the 11 mutants showed a differential effect in bacteriological characteristics such as cell growth, biofilm formation and response to H2O2 stress, revealing the complexity of action mode of these antagonism-related genes. However, a significant reduction of cell motility was observed in the 11 mutants compared to the wild type. Therefore, it can be inferred that the antifungal mechanism of the 10 above-mentioned genes may be, at least partially, due to the weakness of cell motility. Overall, the result of this study will be helpful for us to understand the biocontrol mechanism of this bacterium.

Advancements in Plant and Microbe-Based Synthesis of Metallic Nanoparticles and Their Antimicrobial Activity against Plant Pathogens.

A large number of metallic nanoparticles have been successfully synthesized by using different plant extracts and microbes including bacteria, fungi viruses and microalgae. Some of these metallic nanoparticles showed strong antimicrobial activities against phytopathogens. Here, we summarized these green-synthesized nanoparticles from plants and microbes and their applications in the control of plant pathogens. We also discussed the potential deleterious effects of the metallic nanoparticles on plants and beneficial microbial communities associated with plants. Overall, this review calls for attention regarding the use of green-synthesized metallic nanoparticles in controlling plant diseases and clarification of the risks to plants, plant-associated microbial communities, and environments before using them in agriculture.

Green-Synthesization of Silver Nanoparticles Using Endophytic Bacteria Isolated from Garlic and Its Antifungal Activity against Wheat Fusarium Head Blight Pathogen Fusarium Graminearum.

Nanoparticles are expected to play a vital role in the management of future plant diseases, and they are expected to provide an environmentally friendly alternative to traditional synthetic fungicides. In the present study, silver nanoparticles (AgNPs) were green synthesized through the mediation by using the endophytic bacterium Pseudomonas poae strain CO, which was isolated from garlic plants (Allium sativum). Following a confirmation analysis that used UV-Vis, we examined the in vitro antifungal activity of the biosynthesized AgNPs with the size of 19.8-44.9 nm, which showed strong inhibition in the mycelium growth, spore germination, the length of the germ tubes, and the mycotoxin production of the wheat Fusarium head blight pathogen Fusarium graminearum. Furthermore, the microscopic examination showed that the morphological of mycelia had deformities and collapsed when treated with AgNPs, causing DNA and proteins to leak outside cells. The biosynthesized AgNPs with strong antifungal activity were further characterized based on analyses of X-ray diffraction, transmission electron microscopy, scanning electron microscopy, EDS profiles, and Fourier transform infrared spectroscopy. Overall, the results from this study clearly indicate that the biosynthesized AgNPs may have a great potential in protecting wheat from fungal infection.

Correction: A handy method to remove bacterial contamination from fungal cultures.

[This corrects the article DOI: 10.1371/journal.pone.0224635.].

A Rapid Survey of Avirulence Genes in Field Isolates of Magnaporthe oryzae.

Magnaporthe oryzae is the causal agent for the devastating disease rice blast. The avirulence (AVR) genes in M. oryzae are required to initiate robust disease resistance mediated by the corresponding resistance (R) genes in rice. Therefore, monitoring pathogen AVR genes is important to predict the stability of R gene-mediated blast resistance. In the present study, we analyzed the DNA sequence dynamics of five AVR genes, namely, AVR-Pita1, AVR-Pik, AVR-Pizt, AVR-Pia, and AVR-Pii, in field isolates of M. oryzae in order to understand the effectiveness of the R genes, Pi-ta, Pi-k, Pi-zt, Pia, and Pii in the Southern U.S. rice growing region. Genomic DNA of 258 blast isolates collected from commercial fields of the Southern UNITED STATES during 1975-2009 were subjected to PCR amplification with AVR gene-specific PCR markers. PCR products were obtained from 232 isolates. The absence of PCR products in the remaining 26 isolates suggests that these isolates do not contain the tested AVR genes. Amplified PCR products were subsequently gel purified and sequenced. Based on the presence or absence of the five AVR genes, 232 field isolates were classified into 10 haplotype groups. The results revealed that 174 isolates of M. oryzae carried AVR-Pita1, 225 isolates carried AVR-Pizt, 44 isolates carried AVR-Pik, 3 isolates carried AVR-Pia, and one isolate carried AVR-Pii. AVR-Pita1 was highly variable, and 40 AVR-Pita1 haplotypes were identified in avirulent isolates. AVR-Pik had four nucleotide sequence site changes resulting in amino acid substitutions, whereas three other AVR genes, AVR-Pizt, AVR-Pia, and AVR-Pii, were relatively stable. Two AVR genes, AVR-Pik and AVR-Pizt, were found to exist in relatively larger proportions of the tested field isolates, which suggested that their corresponding R genes Pi-k and Pi-zt can be deployed in preventing blast disease in the Southern UNITED STATES in addition to Pi-ta. This study demonstrates that continued AVR gene monitoring in the pathogen population is critical for ensuring the effectiveness of deployed blast R genes in commercial rice fields.