The Synthesis of Pentyl Leaf Volatiles and Their Role in Resistance to Anthracnose Leaf Blight

Volatiles are important airborne chemical messengers that facilitate plant adaptation to a variety of environmental challenges. Lipoxygenases (LOXs) produce a bouquet of non-volatile and volatile oxylipins, including C6 green leaf volatiles (GLVs), which are involved in a litany of plant physiological processes. GLVs are emitted by a diverse array of plant species, and are the best-known group of LOX-derived volatiles. Five-carbon pentyl leaf volatiles (PLVs) represent another widely emitted group of LOX-derived volatiles that share structural similarity to GLVs, however, relatively little is known about their biosynthesis or biological activity. In this study, we utilized PLV-deficient mutants of maize and Arabidopsis and exogenous PLV applications to elucidate the biosynthetic order of individual PLVs. We further measured PLVs and GLVs after tissue disruption of leaves by two popular methods of volatile elicitation, wounding and freeze-thawing. Freeze-thawing distorted the volatile metabolism of both GLVs and PLVs relative to wounding, though this distortion differed between the two groups of volatiles. These results suggest that despite the structural similarity of these two volatile groups, they are differentially metabolized. Collectively, these results have allowed us to produce the most robust PLV pathway to date. To better elucidate the biological activity of PLVs, we show that PLVs induce maize resistance to the anthracnose pathogen, Colletotrichum graminicola, the effect opposite to that conferred by GLVs. Further analysis of PLV-treated and infected maize leaves revealed that PLV-mediated resistance is associated with early increases of oxylipin α- and γ-ketols, and later increases of oxylipin ketotrienes, hydroxytrienes, and trihydroxydienes. Ultimately, this study has produced the most up-to-date pathway for PLV synthesis, and reveals that PLVs can facilitate pathogen resistance through induction of select oxylipins.

Raman-Based Diagnostics of Stalk Rot Disease of Maize Caused by Colletotrichum graminicola

Stalk rot caused by Colletotrichum graminicola is a disease of worldwide importance. Stalk rot is difficult to detect at the early stages of infection because the fungus colonizes the tissues inside the maize stem. Current diagnostic methods are time-consuming, laborious, and destructive to the stem tissue. We utilized Raman spectroscopy to follow the development of stalk rot in three different maize genotypes grown either in the field or the greenhouse. We then used the acquired spectra to calibrate statistical models to differentiate amongst the different disease timepoints and the genotypes themselves. This non-invasive spectroscopic method enabled high-accuracy identification of stalk rot based on both stalk and leaf spectra. We additionally found that leaf spectra were favorable for identifying maize by genotype. Finally, we identified Raman bands that showed correlation with the sizes of stalk rot-associated lesions in the stems. We demonstrated that Raman spectroscopy is a viable tool for detection of stalk rot disease, as well as potent for the differentiation of maize genotypes.

Transcriptomics differentiate two novel bioactive strains of Paenibacillus sp. isolated from the perennial ryegrass seed microbiome

Paenibacillus species are Gram-positive bacteria that have been isolated from a diverse array of plant species and soils, with some species exhibiting plant growth-promoting (PGP) activities. Here we report two strains (S02 and S25) of a novel Paenibacillus sp. that were isolated from perennial ryegrass (Lolium perenne) seeds. Comparative genomics analyses showed this novel species was closely related to P. polymyxa. Genomic analyses revealed that strains S02 and S25 possess PGP genes associated with biological nitrogen fixation, phosphate solubilisation and assimilation, as well as auxin production and transportation. Moreover, secondary metabolite gene cluster analyses identified 13 clusters that are shared by both strains and three clusters unique to S25. In vitro assays demonstrated strong bioprotection activity against phytopathogens (Colletotrichum graminicola and Fusarium verticillioides), particularly for strain S02. A transcriptomics analysis evaluating nitrogen fixation activity showed both strains carry an expressed nif operon, but strain S02 was more active than strain S25 in nitrogen-free media. Another transcriptomics analysis evaluating the interaction of strains with F. verticillioides showed strain S02 had increased expression of core genes of secondary metabolite clusters (fusaricidin, paenilan, tridecaptin and polymyxin) when F. verticillioides was present and absent, compared to S25. Such bioactivities make strain S02 a promising candidate to be developed as a combined biofertiliser/bioprotectant.

Tracking Fungal Growth: Establishment of Arp1 as a Marker for Polarity Establishment and Active Hyphal Growth in Filamentous Ascomycetes

Polar growth is a key characteristic of all filamentous fungi. It allows these eukaryotes to not only effectively explore organic matter but also interact within its own colony, mating partners, and hosts. Therefore, a detailed understanding of the dynamics in polar growth establishment and maintenance is crucial for several fields of fungal research. We developed a new marker protein, the actin-related protein 1 (Arp1) fused to red and green fluorescent proteins, which allows for the tracking of polar axis establishment and active hyphal growth in microscopy approaches. To exclude a probable redundancy with known polarity markers, we compared the localizations of the Spitzenkörper (SPK) and Arp1 using an FM4-64 staining approach. As we show in applications with the coprophilous fungus Sordaria macrospora and the hemibiotrophic plant pathogen Colletotrichum graminicola, the monitoring of Arp1 can be used for detailed studies of hyphal growth dynamics and ascospore germination, the interpretation of chemotropic growth processes, and the tracking of elongating penetration pegs into plant material. Since the Arp1 marker showed the same dynamics in both fungi tested, we believe this marker can be broadly applied in fungal research to study the manifold polar growth processes determining fungal life.

Novel Paenibacillus sp. Strains From the Perennial Ryegrass Seed Microbiome Reveal Bioprotectant and Biofertiliser Activity - Differentiating Similar Strains via Genomics and Transcriptomics

Paenibacillus species are Gram-positive bacteria that have been isolated from a diverse array of plant species and soils, with some species exhibiting plant growth-promoting (PGP) activities. Here we report a novel Paenibacillus sp. (strains S02 and S25) that was isolated from perennial ryegrass (Lolium perenne) seeds. Comparative genomics analyses showed this novel species was closely related to P. polymyxa. Genomic analyses revealed that strains S02 and S25 possess PGP genes associated with biological nitrogen fixation, phosphate solubilisation and assimilation, as well as auxin production and transportation. Moreover, secondary metabolite gene cluster analyses identified 13 clusters that are shared by both strains and three clusters unique to S25. In vitro assays demonstrated strong bioprotection activity against phytopathogens (Colletotrichum graminicola and Fusarium verticillioides), particularly for strain S02. A transcriptomics analysis evaluating nitrogen fixation activity showed both strains carry an expressed nif operon, but strain S02 was more active than strain S25 in nitrogen-free media. Another transcriptomics analysis evaluating the interaction of strains with F. verticillioides showed strain S02 had increased expression of core genes of secondary metabolite clusters (fusaricidin, paenilan, tridecaptin and polymyxin) when F. verticillioides was present and absent, compared to S25. Such bioactivities make strain S02 a promising candidate to be developed as a combined biofertiliser/bioprotectant.

Genome-wide identification of F-box proteins in Macrophomina phaseolina and comparison with other fungus

BackgroundIn fungi, like other eukaryotes, protein turnover is an important cellular process for the controlling of various cellular functions. The ubiquitin-proteasome pathway degrades some selected intracellular proteins and F-box proteins are one of the important components controlling protein degradation. F-box proteins are well studied in different model plants however, their functions in the fungi are not clear yet. This study aimed to identify the genes involved in protein degradation for disease development in theMacrophomina phaseolinafungus.ResultsIn this research,in silicostudies were done to understand the distribution of F-box proteins in pathogenic fungi includingMacrophomina phaseolinafungus. Genome-wide analysis indicates thatM. phaseolinafungus contained thirty-one F-box proteins throughout its chromosomes. In addition, there are 17, 37, 16, and 21 F-box proteins have been identified fromPuccinia graminis, Colletotrichum graminicola, Ustilago maydis, andPhytophthora infestans, respectively. Analyses revealed that selective fungal genomes contain several additional functional domains along with F-box domain. Sequence alignment showed the substitution of amino acid in several F-box proteins; however, gene duplication was not found among these proteins. Phylogenetic analysis revealed that F-box proteins having similar functional domain was highly diverse form each other showing the possibility of various function. Analysis also found that MPH_00568 and MPH_05531 were closely related to rice blast fungus F-box protein MGG_00768 and MGG_13065, respectively, may play an important role for blast disease development.ConclusionThis genome-wide analysis of F-box proteins will be useful for characterization of candidate F-box proteins to understand the molecular mechanisms leading to disease development ofM. phaseolinain the host plants.

Seed-Borne Fungal Diseases of Maize (Zea mays L.): A Review

Introduction: Maize (Zea mays) is one of the most important cereal crops. It is ranked as 3rd after wheat and rice. Due to its wide adaptability, diversified uses, and low production costs, it has great potential as a cereal crop. In the case of yield losses, various factors are involved. The fungal diseases of maize play a significant role in the reduction of both quantity as well as the quality of maize. Review Results: At the seedling stage, maize suffers from numerous diseases and many of them are seed-borne diseases. Anthracnose stalk rot (Colletotrichum graminicola), Charcoal rot of maize (Macrophomina phaseolina), Crazy top downy mildew disease (Sclerophthora macrospora), Corn grey leaf spot disease (Cercospora zeae-maydis), Aspergillus ear and kernel rot (Aspergillus flavus), Corn smut (Ustilago maydis), Southern corn leaf blight disease (Bipolaris maydis) etc. are important among these diseases.Chemical control of seed-borne pathogens of maize is rather difficult to achieve as a reasonably good. Due to the hazardous environmental effects of chemicals, the Integrated Management of the seed-borne fungal pathogens of corn is mostly preferred. The distribution, disease cycle, symptoms of the damage, effects of environmental factors, economical importance of disease, and integrated disease management options of major seed-borne fungal pathogens of maize have been reviewed in this review article from various currently available sources.

First Report of Colletotrichum sublineola causing Anthracnose on Sorghum bicolor in South Korea

Sorghum (Sorghum bicolor (L.) Moench) is one of the top five cereal crops in the world, but the cultivation area in Korea is estimated to be about 3,000 ha (MIFFAF, 2012). In August 2014, anthracnose symptoms on sorghum leaves were observed in two fields in Yecheon (36.62°, 128.41°) and Youngwol (37.20°, 128.49°), South Korea. Symptoms on leaves were brownish red irregular lesions with yellow and tan borders. Some darkened conidiomata and setae were observed on the lesions of infected leaves. Approximately 20% of sorghum plants (cv. Hwanggeumchal) were affected in each field with an area of about 0.1 ha. Fragments of diseased infected leaves were surface sterilized with 1% NaOCl for 30sec. The pieces were placed on water agar and incubated at 25°C for 7days. Two isolates were obtained through single sporing and cultured on synthetic nutrient poor agar at 25°C for 14days. Conidia (n=30) of YN1458 isolate were falcate and measured 22.0 to 32.7 × 4.2 to 6.4 µm. Brown to black setae (n=20) had 1-3 septa, with tapering acute apices and 53.7 to 95.2 × 4.7 to 7.8 µm in size. Appressoria (n=30) were dark brown, usually irregular and 10.5 to 16.9 × 8.6 to 13.6 µm in size. Colonies on PDA produced salmon spore masses in the center of the colony, and whitish grey to dark color in reverse. The morphological characteristics of two isolates were similar. Based on morphology, two isolates were tentatively identified as Colletotrichum graminicola species complex (Cannon et al. 2012; Crouch and Tomaso-Peterson 2012). To clarify taxonomic placement, DNA extracted from mycelia of the two isolates was PCR amplified and sequenced targeting internal transcribed spacer (ITS) regions of rDNA, actin (ACT), chitin synthase 1(CHS-1), and beta-tubulin (TUB) genes (Weir et al. 2012). The sequences of the above four loci of YN1458 and YN1728 were deposited in GenBank with accession numbers KT351801, KT351802 (ITS); KY769869, KY69870 (ACT); KY769871, KY769872 (CHS-1); and KY769873, KY769874 (TUB), respectively. The sequencing results of two isolates showed 99.6% (ITS), 99.6% (ACT of YN1458), 100% (ACT of YN1728), 100% (CHS-1), 100% (TUB of YN1458) and 99.8% (TUB of YN1728) similarity with C. sublineola CBS 131301 (JQ005771, JQ005834, JQ005792, and JQ005855) by BLASTn. Based on the morphological characteristics and multigene sequence analysis, the two isolates were identified as C. sublineola. Pathogenicity of two isolates was confirmed by spraying conidial suspensions (106 conidia/mL) on leaves of 3-week-old sorghum seedlings (cv. Hwanggeumchal) using a pot assay (5 plants per isolate). The same number of seedlings were sprayed with sterile distilled water and served as controls. All plants were maintained in a greenhouse at 25/32°C with natural light. After one week, symptoms similar to those in the field were observed on the leaves inoculated with the pathogen, but not on the control leaves. Colletotrichum sublineola was consistently re-isolated from the inoculated leaves showing anthracnose symptoms and the pathogen identity was confirmed by observing morphological characteristics. So far, C. graminicola was known as the only causal agent pathogen of sorghum anthracnose in South Korea (KSPP, 2009). To our knowledge, this is the first report of C. sublineola causing anthracnose on sorghum in South Korea. Although sorghum is a small-scale crop in South Korea, it is necessary to study the biological and pathogenic characteristics of C. sublineola for effective control of sorghum anthracnose.