Applications of Pythium- and Phytophthora-produced volatiles in plant disease control.

Volatile organic compounds (VOCs) mediate biological interactions and are produced by Pythium and Phytophthora species. These VOCs are biotechnologically relevant because the genera include important plant pathogens, whereby VOCs can aid in disease detection, and biological control agents, whereby VOCs contribute to disease control. Studies on VOC production, identification, and characterization of individual VOCs produced by Pythium and Phytophthora species are reviewed. VOCs detected in plants infected with Phytophthora species are also reviewed as potentially oomycete-derived VOCs. The Pythium- and Phytophthora-produced VOCs are compared with other microorganisms, and the main effects of these VOCs on microbial inhibition and plant-mediated effects are reviewed. These effects are summarized from direct demonstration studies and inferences based on the known functions of the identified Pythium- and Phytophthora-produced VOCs. There are two main applications of VOCs to plant disease control: the use of VOCs to detect pathogenic Pythium and Phytophthora species, e.g., e-nose detecting systems, and the use of VOC-producing biological control agents, e.g., Pythium oligandrum. Future research could understand how the VOCs are produced to engineer VOC levels in strains, analyze more oomycete species and strains, accurately quantify the VOCs produced, and exploit recent developments in analytical chemistry technology. KEY POINTS: • Compiled inventory of volatiles produced by Phytophthora and Pythium species • Volatilomes contain microbe-inhibiting and plant growth-promoting compounds • Volatile potential in disease detection and control supports analyzing more species.

Volatile Organic Compounds from Pythium oligandrum Play a Role in Its Parasitism on Plant-Pathogenic Pythium myriotylum.

The oomycete Pythium oligandrum is a soil-inhabiting parasite and predator of both fungi and oomycetes, and uses hydrolytic enzymes extensively to penetrate and hydrolyze its host or prey. Other mechanisms have been studied less, and we investigated the contribution of P. oligandrum-produced volatile organic compounds (VOCs) to parasitism. The growth-inhibiting activity of P. oligandrum VOCs was tested on Pythium myriotylum-a host or prey of P. oligandrum-coupled with electron microscopy, and biochemical and transcriptomic analyses. The P. oligandrum-produced VOCs reduced P. myriotylum growth by 80% and zoospore levels by 60%. Gas chromatography-mass spectrometry (GC-MS) identified 23 VOCs, and methyl heptenone, d-limonene, 2-undecanone, and 1-octanal were potent inhibitors of P. myriotylum growth and led to increased production of reactive oxygen species at a concentration that did not inhibit P. oligandrum growth. Exposure to the P. oligandrum VOCs led to shrinkage of P. myriotylum hyphae and lysis of the cellular membranes and organelles. Transcriptomics of P. myriotylum exposed to the P. oligandrum VOCs at increasing levels of growth inhibition initially showed a strong upregulation of putative detoxification-related genes that was not maintained later. The inhibition of P. myriotylum growth continued immediately after the exposure to the VOCs was discontinued and led to the reduced infection of its plant hosts. The VOCs produced by P. oligandrum could be another factor alongside hydrolytic enzymes contributing to its ecological role as a microbial parasite in particular ecological niches such as in soil, and may also contribute to the biocontrol of diseases using P. oligandrum commercial preparations. IMPORTANCE Microbe-microbe interactions in nature are multifaceted, with multiple mechanisms of action, and are crucial to how plants interact with microbes. Volatile organic compounds (VOCs) have diverse functions, including contributing to parasitism in ecological interactions and potential applications in biocontrol. The microbial parasite P. oligandrum is well known for using hydrolytic enzymes as part of its parasitism. We found that P. oligandrum VOCs reduced the growth of, and caused major damage to, the hyphae of P. myriotylum (a host or prey of P. oligandrum). Transcriptomic analyses of P. myriotylum exposed to the VOCs revealed the upregulation of genes potentially involved in an attempt to detoxify the VOCs. The inhibitory effects of the VOCs had a knock-on effect by reducing the virulence of P. myriotylum toward its plant hosts. The P. oligandrum VOCs could contribute to its ecological role as a microbial parasite. The VOCs analyzed here may also contribute to the biocontrol of diseases using P. oligandrum commercial preparations.

Pythium myriotylum Is Recovered Most Frequently from Pythium Soft Rot-Infected Ginger Rhizomes in China.

Pythium soft rot is a major soilborne disease of crops such as ginger (Zingiber officinale). Our objective was to identify which Pythium species were associated with Pythium soft rot of ginger in China, where approximately 20% of global ginger production is located. Oomycetes infecting ginger rhizomes from seven provinces were investigated using two molecular markers, the internal transcribed spacer, and cytochrome c oxidase subunit II (CoxII). In total, 81 isolates were recovered; approximately 95% of the isolates were identified as Pythium myriotylum, and the other isolates were identified as either P. aphanidermatum or P. graminicola. Notably, the P. myriotylum isolates from China did not contain the single nucleotide polymorphism in the CoxII sequence found previously in the P. myriotylum isolates infecting ginger in Australia. A subset of 36 isolates was analyzed repeatedly by temperature-dependent growth, severity of disease on ginger plants, and aggressiveness of colonization on ginger rhizome sticks. In the pathogenicity assays, 32 of 36 isolates were able to significantly infect and cause severe disease symptoms on the ginger plants. A range of temperature-dependent growth, disease severity, and aggressiveness in colonization was found, with a significant moderate positive correlation between growth and aggressiveness of colonization of the ginger sticks. This study identified P. myriotylum as the major oomycete pathogen in China from infected ginger rhizomes and suggested that P. myriotylum should be a key target to control soft rot of ginger disease.

Cryptic Species Identification and Composition of Bemisia tabaci (Hemiptera: Aleyrodidae) Complex in Henan Province, China.

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a cryptic species complex, causing significant crop losses in China during the last decade. Although knowledge of cryptic species composition and dynamics within B. tabaci complex is critical for developing sustainable pest management strategies, limited information is available on this pest in the Henan province of China. A systematic survey of the cryptic species composition and distribution of B. tabaci complex in different locations of Henan province was conducted in 2012. The results of RAPD-PCR and the gene for the mitochondrial cytochrome oxidase subunit-1 (mtCOI) based phylogenetic relationships established using Bayesian method indicated there were four known cryptic species MEAM1, MED, Asia II 3, Asia II 9 and a new cryptic species named China 6 in Henan province. In the survey, the invasive cryptic species MED and MEAM1 were found to be predominant with wide spread distribution across the surveyed regions. On the contrary, the indigenous B. tabaci cryptic species including Asia II 3, Asia II 9 and China 6 remained with low prevalence in some surveyed regions. Cryptic species MEAM1 and MED have not completely displaced the native B. tabaci in Henan province. This current study for the first time unifies our knowledge of the diversity and distribution of B. tabaci across Henan province of China.

Identification and characterization of two phospholipid hydroperoxide glutathione peroxidase genes from the Mediterranean species of the whitefly Bemisia tabaci complex.

Phospholipid hydroperoxide glutathione peroxidases (PHGPXs) are essential enzymes of the cellular antioxidant defense system during insect-plant interactions. However, little attention has been devoted to the functional characterization of PHGXPs in the whitefly Bemisia tabaci. Here, we report the identification and characterization of two PHGPX genes, designated as BtQ-PHGPX1 and BtQ-PHGPX2 from the Mediterranean species of the B. tabaci complex. Sequence analysis indicated that the length of BtQ-PHGPX1 is of 942 bp with a 729 bp open-reading frame (ORF) encoding 242 amino acids, and BtQ-PHGPX2 is of 699 bp with a 567 bp ORF encoding 188 amino acids. Sequence alignment analysis showed that BtQ-PHGPX1 and BtQ-PHGPX2 shared high similarity with other known PHGPXs. The NVASXCGXT, FPCNQFXXQEPG, and IKWNFXKFLV surrounded the reactive cysteine, glutamine, and tryptophan residues, respectively. Recombinant BtQ-PHGPX1 and BtQ-PHGPX2 were overexpressed in Escherichia coli and purified. quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis with whiteflies of different development stages showed that the mRNA levels of BtQ-PHGPX2 were significantly higher in larvae than in other stages. The mRNA levels of BtQ-PHGPX2 were significantly higher than BtQ-PHGPX1 during all the developmental stages. The mRNA levels of BtQ-PHGPX1 and BtQ-PHGPX2 in female adults were relatively higher than in male adults. The expression of BtQ-PHGPX1 and BtQ-PHGPX2 was induced by the insecticide imidacloprid. These results suggest that BtQ-PHGPX1 and BtQ-PHGPX2 may participate in detoxification of oxidative hazards in B. tabaci.

Antibacterial Mechanisms and Antivirulence Activities of Oridonin against Pathogenic Aeromonas hydrophila AS 1.1801.

Aeromonas hydrophila, a Gram-negative bacterium widely found in freshwater environments, acts as a common conditional pathogen affecting humans, livestock, and aquatic animals. In this study, the impact of oridonin, an ent-kaurane diterpenoid compound derived from Rabdosia rubescens, on the virulence factors of A. hydrophila AS 1.1801 and its antibacterial mechanism was elucidated. The minimum inhibitory concentration (MIC) of oridonin against A. hydrophila AS 1.1801 was 100 µg/mL. Oridonin at inhibitory concentrations could significantly increase the electrical conductivity in the supernatant and escalate nucleic acid leakage (p < 0.01). This effect was concomitant with observed distortions in bacterial cells, the formation of cytoplasmic cavities, cellular damage, and pronounced inhibition of protein and nucleic acid synthesis. Additionally, oridonin at inhibitory levels exhibited a noteworthy suppressive impact on A. hydrophila AS 1.1801 across biofilm formation, motility, hemolytic activity, lipase activity, and protease activity (p < 0.05), demonstrating a dose-dependent enhancement. qRT-PCR analysis showed that the gene expression of luxR, qseB and omp were significantly downregulated after oridonin treatment in A. hydrophila AS 1.1801 (p < 0.05). Our results indicated that oridonin possessed significant antibacterial and anti-virulence effects on A. hydrophila AS 1.1801.

Prokaryotic expression, purification, and the in vitro and in vivo protection study of dehydrin WDHN2 from Triticum aestivum.

Dehydrins proteins accumulate and play important protective roles in most plants during abiotic stresses. The objective of this study was to characterize a YSK2-type dehydrin gene, WDHN2, isolated from Triticum aestivum previously. In this work, wheat dehydrin WDHN2 was expressed in Escherichia coli and purified by immobilized metal affinity chromatography, which exhibited as a single band by sodium dodecyl sulfonate polyacrylamide gel electrophoresis and western blotting. We show that WDHN2 is capable of alleviating lactate dehydrogenase inactivation from heat and desiccation in vitro enzyme activity protection assay. In vivo assay of Escherichia coli viability demonstrates the enhancement of cell survival by the overexpression of WDHN2. The protein aggregation prevention assay explores that WDHN2 has a broad protective effect on the cellular proteome. The results show that WDHN2 is mainly accumulated in the nucleus and cytosol, suggesting that this dehydrin may exert its function in both cellular compartments. Our data suggest that WDHN2 acts as a chaperone molecular in vivo.

Growth, Enzymatic, and Transcriptomic Analysis of xyr1 Deletion Reveals a Major Regulator of Plant Biomass-Degrading Enzymes in Trichoderma harzianum.

The regulation of plant biomass degradation by fungi is critical to the carbon cycle, and applications in bioproducts and biocontrol. Trichoderma harzianum is an important plant biomass degrader, enzyme producer, and biocontrol agent, but few putative major transcriptional regulators have been deleted in this species. The T. harzianum ortholog of the transcriptional activator XYR1/XlnR/XLR-1 was deleted, and the mutant strains were analyzed through growth profiling, enzymatic activities, and transcriptomics on cellulose. From plate cultures, the Δxyr1 mutant had reduced growth on D-xylose, xylan, and cellulose, and from shake-flask cultures with cellulose, the Δxyr1 mutant had ~90% lower ß-glucosidase activity, and no detectable ß-xylosidase or cellulase activity. The comparison of the transcriptomes from 18 h shake-flask cultures on D-fructose, without a carbon source, and cellulose, showed major effects of XYR1 deletion whereby the Δxyr1 mutant on cellulose was transcriptionally most similar to the cultures without a carbon source. The cellulose induced 43 plant biomass-degrading CAZymes including xylanases as well as cellulases, and most of these had massively lower expression in the Δxyr1 mutant. The expression of a subset of carbon catabolic enzymes, other transcription factors, and sugar transporters was also lower in the Δxyr1 mutant on cellulose. In summary, T. harzianum XYR1 is the master regulator of cellulases and xylanases, as well as regulating carbon catabolic enzymes.

Volatile Organic Compounds Emitted by the Biocontrol Agent Pythium oligandrum Contribute to Ginger Plant Growth and Disease Resistance.

The oomycete Pythium oligandrum is a potential biocontrol agent to control a wide range of fungal and oomycete-caused diseases, such as Pythium myriotylum-caused rhizome rot in ginger, leading to reduced yields and compromised quality. Previously, P. oligandrum has been studied for its plant growth-promoting potential by auxin production and induction of disease resistance by elicitors such as oligandrin. Volatile organic compounds (VOCs) play beneficial roles in sustainable agriculture by enhancing plant growth and resistance. We investigated the contribution of P. oligandrum-produced VOCs on plant growth and disease suppression by initially using Nicotiana benthamiana plants for screening. P. oligandrum VOCs significantly enhanced tobacco seedling and plant biomass contents. Screening of the individual VOCs showed that 3-octanone and hexadecane promoted the growth of tobacco seedlings. The total VOCs from P. oligandrum also enhanced the shoot and root growth of ginger plants. Transcriptomic analysis showed a higher expression of genes related to plant growth hormones and stress responses in the leaves of ginger plants exposed to P. oligandrum VOCs. The concentrations of plant growth hormones such as auxin, zeatin, and gibberellic acid were higher in the leaves of ginger plants exposed to P. oligandrum VOCs. In a ginger disease biocontrol assay, the VOC-exposed ginger plants infected with P. myriotylum had lower levels of disease severity. We conclude that this study contributes to understanding the growth-promoting mechanisms of P. oligandrum on ginger and tobacco, priming of ginger plants against various stresses, and the mechanisms of action of P. oligandrum as a biocontrol agent. IMPORTANCE Plant growth promotion plays a vital role in enhancing production of agricultural crops, and Pythium oligandrum is known for its plant growth-promoting potential through production of auxins and induction of resistance by elicitors. This study highlights the significance of P. oligandrum-produced VOCs in plant growth promotion and disease resistance. Transcriptomic analyses of leaves of ginger plants exposed to P. oligandrum VOCs revealed the upregulation of genes involved in plant growth hormone signaling and stress responses. Moreover, the concentration of growth hormones significantly increased in P. oligandrum VOC-exposed ginger plants. Additionally, the disease severity was reduced in P. myriotylum-infected ginger plants exposed to P. oligandrum VOCs. In ginger, P. myriotylum-caused rhizome rot disease results in severe losses, and biocontrol has a role as part of an integrated pest management strategy for rhizome rot disease. Overall, growth enhancement and disease reduction in plants exposed to P. oligandrum-produced VOCs contribute to its role as a biocontrol agent.