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Rice sheath blight, which is caused by Rhizoctonia solani, is a devastating disease in many areas of the world where rice is cultivated. The role of polygalacturonase (PG) in the infection process of R. solani was investigated in the present study by cloning and investigating the function of a new PG-encoding gene, which was designated RsPG2. Bioinformatics showed that RsPG2 consisted of a 1,633 bp open reading frame (ORF) with six introns; the deduced protein contained 436 amino acids with a predicted mass of 45.88 kDa after cleavage of the predicted 17-amino acid signal sequence. Culture supernatants obtained from Pichia pastoris producing recombinant RsPG2 degraded polygalacturonic acid in vitro, thus confirming that RsPG2 encodes a PG gene. Purified recombinant RsPG2 also degraded rice tissue and elicited obvious necrotic symptoms 48 h after inoculation. Real-time qRT-PCR indicated that the expression of RsPG2 was strongly induced during the infection of rice by R. solani. These data unequivocally demonstrate that RsPG2 plays an important role in R. solani infection and provide further insights for elucidating the function of pg genes in the virulence of R. solani.

Citrus mandarin (Citrus reticulata) cultivar Shatangju has great economic importance in southern China because of its delicious fruit and medicinal peel. During surveys conducted in 2012 and 2013, stem rot and plant death were observed on 2- to 3-year-old Shatangju grafted seedlings in 21 nurseries located in three regions (Yangjiang City, Sihui City and Deqing County) of Guangdong Province, China. The aims of this study were to characterize the symptoms of seedling stem rot and death; evaluate the incidence and loss; identify the causal agent and evaluate its pathogenicity. The disease was present in all surveyed nurseries but incidence varied with location. A Pythium-like oomycete was consistently obtained from symptomatic plants collected in the nurseries surveyed. On the basis of morphological and cultural characteristics and combined phylogenetic analysis of three DNA sequences including the internal transcribed spacer region (ITS), mitochondrial CoxI and CoxII genes, 21 of the isolates obtained were identified as belonging to the species Phytopythium helicoides. The isolates were pathogenic to the scions but not rootstocks of Shatangju mandarin seedlings cultivated in a controlled greenhouse and symptoms identical to that observed in the field were reproduced. The pathogen was re-isolated consistently from the inoculated plants, confirming Koch’s postulates. Seedling stem rot and eventual death caused by P. helicoides represents a serious threat to Shatangju mandarin nursery productivity and longevity in southern China.

Peptides and small molecules produced by both the plant pathogen Phytophthora and host plants in the apoplastic space mediate the relationship between the interplaying organisms. Various Phytophthora apoplastic effectors, including small cysteine-rich (SCR) secretory proteins, have been identified, but their roles during interaction remain to be determined. Here, we identified an SCR effector encoded by scr96, one of three novel genes encoding SCR proteins in P. cactorum with similarity to the P. cactorum phytotoxic protein PcF. Together with the other two genes, scr96 was transcriptionally induced throughout the developmental and infection stages of the pathogen. These genes triggered plant cell death (PCD) in the Solanaceae, including Nicotiana benthamiana and tomato. The scr96 gene did not show single nucleotide polymorphisms in a collection of P. cactorum isolates from different countries and host plants, suggesting that its role is essential and non-redundant during infection. Homologues of SCR96 were identified only in oomycetes, but not in fungi and other organisms. A stable protoplast transformation protocol was adapted for P. cactorum using green fluorescent protein as a marker. The silencing of scr96 in P. cactorum caused gene-silenced transformants to lose their pathogenicity on host plants and these transformants were significantly more sensitive to oxidative stress. Transient expression of scr96 partially recovered the virulence of gene-silenced transformants on plants. Overall, our results indicate that the P. cactorum scr96 gene encodes an important virulence factor that not only causes PCD in host plants, but is also important for pathogenicity and oxidative stress tolerance.

Bacillus sp. 6256 is a good biocontrol agent against Botrytis cinerea which caused tomato gray mold disease. Strain 6256 was identified as B. amyloliquefaciens by analysis of its partial gyrB gene sequence. To identify and characterize the antimycotic peptides from the culture broth of the bacterium, the antimicrobial substances produced by B. amyloliquefaciens 6256 were isolated by ammonium sulfate precipitation, Superdex 200 gel filtration chromatography and DEAE anion exchange chromatography. The purified compound was designated as P657. The biological activity of P657 was stable at as high as 100 °C for 20 min and in pH value ranged from 5 to 10. The antimycotic compound was resistant to trypsin and proteinase K, and could completely inhibit spore germination of Botrytis cinerea in vitro. MALDI-TOF-MS analysis results showed the presence of fengycins A (C16–C17) and fengycins B (C15–C17) isoforms in P657.

Antifungal metabolites produced by Bacillus sp. W10, which was previously isolated from the tomato rhizosphere, were investigated. Strain W10 was identified as Bacillus amyloliquefaciens by analysis of its 16S rDNA and gyrB gene partial sequences. PCR analysis showed the presence of fenB, sfp, and ituD genes, coding for fengycin, surfactin, and iturin, respectively. A novel small antifungal peptide, designated 5240, produced by this strain was isolated by ammonium sulfate precipitation and Superdex 200 gel filtration chromatography. The 5240 peptide was stable at 100 °C for 20 min and remained active throughout a wide pH range (4–10). The antagonistic activity was not affected by protease K and trypsin. The purified 5240 peptide exhibited a broad inhibitory spectrum against various plant pathogenic fungi and was identified as iturin A (C14-C16). Moreover, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated the presence of fengycin A (C14-C15), fengycin B (C16-C17), and surfactin (C13-C16) isoforms in supernatants from strain W10. These results suggest that B. amyloliquefaciens W10 has significant potential as a biocontrol agent.

Using the reference sequences of pgip genes in GenBank, a fragment of 930 bp covering the open reading frame (ORF) of rice Ospgip1 (Oryza sativa polygalacturonase-inhibiting protein 1) was amplified. The prokaryotic expression product of the gene inhibited the growth of Rhizoctonia solani, the causal agent of rice sheath blight, and reduced its polygalacturonase activity. Bioinformatic analysis showed that OsPGIP1 is a hydrophobic protein with a molecular weight of 32.8 kDa and an isoelectric point (pI) of 7.26. The protein is mainly located in the cell wall of rice, and its signal peptide cleavage site is located between the 17th and 18th amino acids. There are four cysteines in both the N- and C-termini of the deduced protein, which can form three disulfide bonds (between the 56th and 63rd, the 278th and 298th, and the 300th and 308th amino acids). The protein has a typical leucine-rich repeat (LRR) domain, and its secondary structure comprises α-helices, β-sheets and irregular coils. Compared with polygalacturonase-inhibiting proteins (PGIPs) from other plants, the 7th LRR is absent in OsPGIP1. The nine LRRs could form a cleft that might associate with proteins from pathogenic fungi, such as polygalacturonase.

Background Phytophthora cactorum, a hemibiotrophic oomycete pathogen, can cause destructive diseases on numerous crops worldwide, leading to essential economic losses every year. However, little has been known about its molecular pathogenicity mechanisms. To gain insight into its repertoire of effectors, the P. cactorum transcriptome was investigated using Illumina RNA-seq. Results We first demonstrated an in vitro inoculation method that can be used to mimic natural cyst germination on host plants. Over 28 million cDNA reads were obtained for five life cycle stages (mycelium, sporangium, zoospore, cyst and germinating cyst) and de novo assembled into 21,662 unique genes. By comparisons with 11 public databases, 88.99% of the unique genes were annotated, including 15,845 mapped to the gene models of the annotated relative Phytophthora infestans. Using TribeMCL, 5,538 gene families conserved across P. cactorum and other three completely sequenced Phytophthora pathogen species were determined. In silico analyses revealed that 620 P. cactorum effector homologues including 94 RXLR effector candidates matched known or putative virulence genes in other oomycetes. About half of the RXLR effector candidates were predicted to share a conserved structure unit, termed the WY-domain fold. A subset of the effector genes were checked and validated by PCR amplification. Transcriptional experiments indicated that effector genes were differentially expressed during the life cycle and host infection stages of P. cactorum. Ectopic expression in Nicotiana benthamiana revealed that RXLR, elicitin and NLP effectors can trigger plant cell death. These effectors are highly conserved across oomycete species. Single nucleotide polymorphisms for RXLR effectors were detected in a collection of P. cactorum isolates from different countries and hosts. Conclusions This study demonstrates the comprehensive sequencing, de novo assembly, and analyses of the transcriptome of P. cactorum life cycle stages. In the absence of genome sequence, transcriptome data is important for infection-related gene discovery in P. cactorum, as demonstrated here for the effector genes. The first look at the transcriptome and effector arsenal of P. cactorum provides valuable data to elucidate the pathogenicity basis of this broad-host-range pathogen. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-980) contains supplementary material, which is available to authorized users.

There was an error in Table 5. A correct version of the error in available here: http://www.plosone.org/corrections/pone.0074588.g005.tif

Red bayberry (Myrica rubra Seib. & Zucc.) has great economic importance in eastern and southern China. However, increasing cultivation of red bayberry has resulted in an increase in diseases such as leaf necrosis. In April 2011, a survey was conducted to identify the causal agents of leaf necrosis of red bayberry (cv. Biqi) in Cixi City, Zhejiang Province. Symptoms began with oval and pale brown lesions (2 mm in diameter) that developed into a round to irregular shape (4 to 12 mm in diameter) with pale brown centers and dark brown borders. After approximately 4 months, necrotic lesions expanded to the leaf tips or margins. Black acervuli developed on lesions at later stages. Leaf tissues were surface sterilized with 0.5% sodium hypochlorite for 3 min and rinsed in sterile water before plating onto potato dextrose agar (PDA). Seven isolates were obtained from four samples from four fields on PDA at 25°C. The colonies were cottony white with filiform edges and produced a honey yellow color into the agar at 7 days. Conidia were produced in ink-like fruiting bodies at 4 days at 25°C on PDA. Conidia were straight or slightly curved, fusiform, and five celled with constrictions at the septa. Conidia ranged from 18.7 to 25.8 × 6.2 to 7.7 μm with hyaline apical and basal cells. Thirteen percent of the apical cells had two and the rest had three hyaline appendages ranging from 11.2 to 26.0 μm long. Basal appendages were hyaline, straight, and varied from 3.6 to 5.8 μm long. The color of three median cells was light to dark brown and demonstrated versicolorous. These morphological characteristics matched those of Pestalotiopsis sydowiana (Bresadola) Sutton (1). The morphological identification of the fungus was confirmed by nucleotide blast analysis of the 5.8S subunit and flanking internal transcribed spacers (ITS1 and ITS2) of rDNA regions (GenBank Accession No. JQ322999), which revealed 100% with those of other P. sydowiana isolates (e.g., GenBank Accession No. FJ478105). Koch's postulates were confirmed with 20 healthy leaves of the same size on three branches of three plants in the field. Leaves were wounded by pressing slightly with sterile needles. Mycelial plugs (5 mm in diameter) obtained from the periphery of 7-day-old cultures were placed onto the wounds and covered with sterile-water-saturated cotton. Wounded leaves treated with sterile agar plugs served as controls. The inoculated leaves were sealed in moist plastic bags for 24 h to establish high humid conditions at 21 to 30°C. After 23 days, symptoms on all inoculated leaves were identical to those described above, whereas noninoculated control leaves did not show any symptoms. The fungus was consistently reisolated from the lesions. To our knowledge, this is the first report of P. sydowiana causing leaf necrosis of M. rubra in China. Results can help to better understand the diseases threatening red bayberry trees and develop effective control strategies for better fruit production. Reference: (1) E. F. Guba. Monograph of Monochaetia and Pestalotia. Harvard University Press, Cambridge, MA, 1961.