Occurrence of Fusarium fujikuroi Causing Fusarium Wilt on Carthusian Pink in China.

Carthusian pink (Dianthus carthusianorum) is native to Europe and is widely grown in China for landscaping. In September 2022, wilting symptoms of carthusian pink were found in Xixia City (33°18'31″ N, 111°29'45″ E), Henan Province, China, with a disease incidence of 65%. Approximately 100 plants were surveyed on the landscaping lawns of the park. Initial symptoms were yellow to brown lesions on the base of stems and leaves. Later, the lesions spread throughout the plants, turning leaves yellow, and leading to root and leaf rot. Eventually, the plants shriveled and died (Figure S1a). Thirty diseased tissues isolated from the roots and leaves were cut into 5×5 mm pieces, which were surface sterilized with 75% ethanol solution for 30 seconds and 1% NaClO solution for 1 minute, rinsed three times in sterilized water, placed on potato dextrose agar (PDA) plates supplemented with 50 µg ml-1 streptomycin, and incubated at 28°C for five days. A total of 25 purified fungal strains with similar phenotypic features were obtained. Three representative strains named OSZ-P1, OSZ-P2, and OSZ-P3 were selected for identification. Fungal colonies developed an abundant aerial mycelium, initially white, which subsequently developed red to purple pigments (Figure S1b). Macroconidia were slender, straight, and measured 12.74 to 49.39 × 2.07 to 4.39 µm (n=50), with two to five septa. Microconidia were clavate and measured 6.31 to 11.61 × 2.15 to 4.02 µm (n=50) (Figure S1c). These morphological characteristics were consistent with Fusarium spp.. The rDNA internal transcribed spacer (ITS), ß-tubulin gene (tub2), translation elongation factor 1-alpha gene (tef1), calmodulin (cmdA), RNA polymerase largest subunit (rpb1), and RNA polymerase II second largest subunit (rpb2) were amplified with primers ITS1/ITS4, BT-2a/BT-2b, EF1/EF2, CL1/CL2A, Fa/G2R, and 5F2/7Cr, respectively, for further identification (Yilmaz et al. 2021, O'Donnell et al. 2022). ITS (OQ726389, OQ726390, OQ726391), tub2 (OQ730191, OQ789645, OQ789646), tef1 (OR088904, OR088905, OR088906), cmdA (OR133730, OR133731, OR133732), rpb1 (OR088907, OR088908, OR133729), and rpb2 (OR133733, OR133734, OR133735) nucleotide sequences of the strains OSZ-P1, OSZ-P2, and OSZ-P3 were submitted to GenBank. BLASTn analysis of OSZ-P1 sequences exhibited 99 to 100% similarity with Fusarium fujikuroi sequences (strains Augusto2, I1.3, and CSV1) CP023096, CP023108, CP023084 of cmdA, CP023089, CP023077 of rpb1, and CP023093, CP023105, CP023081 of rpb2. A Phylogenetic tree was constructed of combined genes (tub2, tef1, cmdA, rpb1, rpb2) of sequences, alongside the sequences of the type strains by the neighbor-joining method. The three strains formed a clade with the type strains CBS257.52 and Augusto2 of F. fujikuroi in phylogenetic trees, being clearly separated from other Fusarium spp. (Figure S2). The morphological features and molecular analyses supported the strains as members of F. fujikuroi. To verify the pathogenicity, aboveground parts of the plants of five healthy six-month-old potted plants were sprayed with 100 µl of conidial suspension per pot (106 conidia ml-1), and five similar plants were sprayed with sterilized water as a control. All plants were placed in a climate incubator at 28°C and 90% relative humidity. Seven days after inoculation, withered and yellowed lesions were observed, similar to the natural lesions (Figure S1e). No symptoms were observed on the control plants. The whole pathogenicity tests were performed thrice. Reisolation resulted in cultures that were morphologically and molecularly identical to the original isolates, fulfilling Koch's postulates. Fusarium wilt disease has been reported on other plants of the genus Dianthus. Vascular wilt on Dianthus caryophyllus (carnation) caused by Fusarium oxysporum is the most destructive disease of carnation crops worldwide (Ardila et al. 2014). Fusarium acuminatum causing Dianthus chinensis root rot and foliage blight has recently been reported in Nanjing, China (Xu et al. 2022). To our knowledge, this is the first report of F. fujikuroi causing Fusarium wilt on carthusian pink worldwide. The host range of F. fujikuroi still needs to be clarified for accurate disease management in the selection of plant species for landscape.

First report of Fusarium falciforme causing root rot of pomegranate (Punica granatum L.) in China.

Pomegranate (Punica granatum L.) is a deciduous shrub or small tree that is native to Iran and Afghanistan. It is also a commercially important fruit tree in China and worldwide. In the summer of 2022, a serious root rot disease occurred in some pomegranate orchards in Xichuan County(32º42´ N, 111º48´ E), Henan Province, China, with an incidence of ~30%. Symptoms included leaf yellowing and wilting, root browning and rotting, and stem-base cracking, eventually leading to defoliation and death. To isolate the causal agent, small pieces (5×5 mm) of diseased root from six trees were surface-sterilized by dipping in 2% NaClO for 8 min followed by 70% ethanol for 15 s, rinsed five times with sterile water, and plated on potato dextrose agar (PDA), then incubated at 28°C in the dark for 5 days. Fifteen pure fungal isolates with the same morphological characteristics were obtained from 24 pieces of roots. All isolates produced white fluffy mycelia. Microconidia were hyaline, oval or reniform, with zero to one septa and dimensions of 7.1 to 19.9 (average 14.5 )× 3.8 to 8.0 (average 5.6) µm (n = 100). Macroconidia were sickle-shaped, one to four septate, and 20.1 to 40.8 (average 26.5) × 4.8 to 8.6 (average 6.5) µm (n = 100). Chlamydospores were spherical, single, in pairs or chains, and 5.6 to 9.8 (average 6.8) µm in diameter (n = 100). Based on the above characteristics, the pathogens were identified as Fusarium sp. (Leslie and Summerell 2006). Genomic DNA was extracted from mycelia of two representative isolates Fs1 and Fs3. The internal transcribed spacer (ITS), translation elongation factor 1-alpha (TEF-1α) and RNA polymerase II second largest subunit (RPB2) sequences were PCR amplified using primer pairs of ITS1/ITS4, EF1/EF2, and RPB2-5f2/RPB2-7cr, RPB2-7cf/RPB2-11ar (O'Donnell et al., 2022), respectively. BLAST analysis showed that the ITS, TEF-1α and RPB2 sequences of isolates Fs1(GenBank accession nos. OK001765, OQ921726 and OQ928396) and Fs3 (GenBank accession nos. OK001771, OQ921727 and OQ928397) showed 99%-100% identity with multiple GenBank sequences of Fusarium falciforme (KY617066, MN064683, KF255514, OQ933361, KY556711 and ON331935). A phylogenetic tree based on concatenated sequences of ITS, TEF-1α and RPB2 using maximum-likelihood analysis revealed that both isolates Fs1 and Fs3 were in the same clade with F. falciforme strains. Based on the morphological and molecular characteristics, the isolates were identified as members of F. falciforme. For pathogenicity testing, conidial suspensions (1×108 spores /mL) of isolates Fs1 and Fs3 were poured onto the roots of healthy pomegranate that had been planted in pots two months previously. Ten plants were inoculated for each isolate. Control plants were drenched with sterile water. After 3 months, inoculated plants developed leaf yellowing and wilting accompanied by root browning and rotting, much like symptoms observed in field plants. The same fungi re-isolated from the experimental plants were confirmed to be F. falciforme by morphology and sequence analysis. This is the first report of F. falciforme causing root rot on pomegranate. F. falciforme is a ubiquitous soil-borne pathogen that causes root rot on multiple plants around the world (Xu F., et al. 2022; Qiu R., et al. 2023). The results of pathogen identification are essential precursors to development of effective control of the disease.

Leaf Spot Disease on Punica granatum Caused by Alternaria alternata in China.

Pomegranate (Punica granatum L.), which is native to central Asia, is considered as one of the most renowned commercial fruit trees in the world. The planting area in China is roughly 120 thousand hectares. In June 2020, symptoms of leaf spot on P. granatum appeared in Nanyang City (32º40´34˝N, 111º44´20˝E), Henan Province, with an incidence rate of 35% in several 3.3-hectare orchards. Initially, the lesions showed as round or subrounded brown spots on affected leaves. The spots then progressively developed into irregular lesions with distinct yellow halos surrounding them. Parts of the lesions were weakly zonate, which finally led to leaf withering and falling off. Diseased tissues were cut into 5×5 mm2 pieces, which were surface sterilized with 75% ethanol solution for 30 s, washed 3 times in sterilized water, and put on potato dextrose agar (PDA) plates supplemented with 50 µg ml-1 streptomycin. A total of 16 purified fungal isolates with similar phenotypic features were obtained. Three randomly chosen isolates SLY11, SLY24, and SLY25 were utilized for the investigation. Fungal colonies on PDA were first white to gray and later mycelium became olive green to blackish brown. To examine the morphological properties of conidia, we utilized potato carrot agar (PCA) culture medium and incubated it at 23°C under a 12-hour light/dark alternation. Conidia were obclavate or spheroidal, dark brown, with 3 to 5 transverse septa and 1 to 4 longitudinal septa. Conidiophores were septate, solitary, and measured 22.7 (±4.64) × 10.6 (±2.15) µm (n=50), with a conical beak length of 0 to 5.5 µm. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-alpha gene (TEF1), ß-tubulin gene (TUB), and glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) were amplified using primer pairs ITS1/ITS4, EF1-728F/EF1-986R, Bt2a/Bt2b, and GDF1/GDF2 from genomic DNA. Sequences were submitted to GenBank with accession numbers OL840230, OL840231, OL840232 for ITS, OL982540, OL982541, OL982542 for TEF1, OL982543, OL982544, OL982545 for TUB, OL862608, OL862609, OL862610 for GAPDH sequences of isolates SLY11, SLY24, and SLY25, respectively. BLASTn analysis of ITS (OL840230), TEF1 (OL982540), TUB (OL982543), GAPDH (OL862608) sequences indicated 100, 99.59, 99.68, and 100% similarity to the sequences of Alternaria alternata strain HC-2 (MT644140), BJFA-1 (MK895958), CS36-5 (KY814630), and ag1 (KP057228) in GenBank. Isolates SLY11, SLY24, and SLY25 formed a clade with the type strains CBS 130265 and CBS 130258 of A. alternata in phylogenetic trees established, clearly seperating from other Alternaria spp. The morphological features and molecular analyses supported the isolates as members of A. alternata. To validate the pathogenicity of the isolates, ten healthy leaves of 3-year-old potted pomegranate trees were utilized for testing and inoculated with conidial suspension (106 conidia ml-1), 20 µl each leaf. Control plants were inoculated with sterilized water. An additional pathogenicity test was repeated on wounded leaves. The inoculated plants were placed at 28°C in a greenhouse (12 h light per day and 90% relative humidity) for 5 days. The pathogenicity testing was conducted three times. Distinct lesions were found on the nonwounded and wounded leaves of inoculated plants after 3 to 5 days. The morphology and ITS sequences of the fungi that were reisolated from each of the inoculated plants were similar to that of the inocula, fulfilling Koch's postulates. Fruit rot of pomegranate induced by A. alternata was not identified in our investigation. A. alternata is reported to induce leaf spot disease on P. granatum in India (Zakir et al. 2009), Israel (Ezra et al. 2010), Spain (Garcia-Jimenez et al. 2014). To our knowledge, this is the first report of A. alternata causing leaf spot disease on P. granatum in China. Severe leaf disease caused by A. alternata can lead to reduced pomegranate yields in the harvest stages. This note will aid in pathogen identification and disease control.

First Report of Leaf Black Spot Caused by Alternaria alternata on Rosemary (Rosmarinus officinalis L.) in China.

Rosemary (Rosmarinus officinalis L.) is an aromatic, evergreen, medicinally important shrub and widely used for cooking, tea, cosmetics as well as medicinal materials. It is grown in many countries including China that had more than 9300 hm2 of commercial cultivation area in 2021. In March 2020, a leaf spot disease sporadic occurred in field rosemarry plants in Nanyang City (32º51´ N, 111º36´ E), Henan Province, China. The disease outbreaked in September with a disease incidence of 57-83%. Symptoms initially appeared as small brown leaf spots that gradually expanded into dark blackbrown irregular lesions. Most of the spots started from the leaf tip or leaf margin, and gradually spread to the leaf base, resulting in heavy defoliation especially on rainy days. Diseased leaf segments (1×3 mm) were surface-sterilized by dipping in 1% sodium hypochlorite for 1 min, rinsed three times with sterile distilled water, and plated on potato dextrose agar, then incubated at 28°C in the dark for 5 days. Twelve fungal isolates with the same morphological characteristics were obtained from nine affected leaves. The fungal colonies were initially white and turned gray brown with flocculent aerial mycelia and a whorled back. Conidia were frequently born in a long chain, with a short beak, brown or light-brown, 13.2 to 48. 7 (average 26.1) × 4.0 to 13.1 (average 8.0) µm in size (n=148) with 0 to 8 transverse and 0 to 3 longitudinal/oblique septa. Phenotypic features of the isolates agreed with those of Alternaria alternata (Simmons et al. 2007). Two isolates Aa1 and Aa2 were randomly selected for molecular and pathogenicity tests. DNA was extracted from mycelia. Partial sequences of internal transcribed spacer (ITS) and translation elongation factor 1-alpha (TEF1-α) were amplified using the primer pairs ITS1/ITS4 and EFI-728F/EFI-986R (Wei et al. 2022), respectively. The GenBank accession nos. were OK036714 and OK036715 for ITS, and ON951980 and ON951981 for TEF1-α of Aa1 and Aa2, respectively, with a maximal identity of greater than 99% to multiple A. alternata strains. In the neighbour joining phylogenetic tree of the amplified ITS and TEF1-α sequences both Aa1 and Aa2 clustered with A. alternata strains, clearly separating them from other Alternaria spp. For pathogenicity test, conidial suspensions (1×106 spores /mL) of Aa1 and Aa2 were separately sprayed on healthy one-year-old rosemary plants (n=3) with their leaves slightly wounded with a sterilized needle. Control plants (n=3) were sprayed with sterile water. Both inoculated and control plants were incubated at 90% RH, 28 °C. After 14 days, all the inoculated leaves showed black brown lesions similar to those on naturally affected field plants, whereas controls remained symptomless. Fungal cultures with the same phenotypic features as the inocula were constantly re-isolated from the infected leaves. A. alternata was reported as pathogen causing foliar necrosis on rosemary in Italy (Perello et al.1995) and leaf spot (or leaf blight) on multiple plant species such as Actaea dahurica (Hai et al. 2022), and Ligustrum japonicum (Wei et al. 2022) in China. This is the first report of A. alternata causing leaf black spot on rosemary in China.

Characterization of a novel chitinolytic Serratia marcescens strain TC-1 with broad insecticidal spectrum.

The Gram-negative rod-shaped bacterium Serratia marcescens is an opportunistic pathogen of many organisms, including insects. We report the identification and optimal in vitro chitinase production conditions of a novel chitinolytic S. marcescens strain TC-1 isolated from a naturally infected white grub (Anomala corpulenta) collected from a peanut field at Nanyang city, Henan province, China. Strain identification was conducted by morphological, physiological, biochemical and molecular analyses. The amplified 16S rRNA gene of TC-1 showed a similarity greater than 99% with multiple strains of S. marcescens. Based on Neighbor-joining phylogenetic tree analysis of bacterial 16S rRNA gene sequences, TC-1 formed a clade with S. marcescens, clearly separated from other Serratia spp. The strain TC-1 showed larvicidal activities against five insect species (A. corpulenta, Plutella xylostella, Spodoptera exigua, Helicoverpa armigera, Bombyx mori) and the nematode Caenorhabditis elegans, but not against S. litura. The operating parameters of chitinase production by TC-1 were optimized by response surface methodology using a three-factor, three-level Box-Behnken experimental design. The effects of three independent variables i.e. colloidal chitin concentration (7-13 g l-1), incubation time (24-72 h) and incubation temperature (24-32 °C) on chitinase production by TC-1 were investigated. A regression model was proposed to correlate the independent variables for an optimal chitinase activity predicted as 20.946 U ml-1, using a combination of colloidal chitin concentration, incubation time and incubation temperature of 9.06 g l-1, 63.83 h and 28.12 °C, respectively. The latter agreed well with a mean chitinase activity of 20.761 ± 0.102 U ml-1 measured in the culture supernatants of TC-1 grown under similar conditions with a colloidal chitin concentration, incubation time and incubation temperature of 9 g l-1, 64 h and 28 °C, respectively. Our study revealed the S. marcescens strain TC-1 with potential as a biocontrol agent of insect pests and nematodes and demonstrated the proposed regression model's potential to guide chitinase production by this strain.

Plant organ- and growth stage-diversity of endophytic bacteria with potential as biofertilisers isolated from wheat (Triticum aestivum L.).

BACKGROUND: Chemical fertilisers are extensively used for crop production, which may cause soil deterioration and water pollution. Endophytic bacteria with plant-growth-promoting (PGP) activities may provide a solution to sustainably improve crop yields, including in-demand staples such as wheat. However, the diversity of the PGP endophytic bacteria in wheat across plant organs and growth stages has not been thoroughly characterised. RESULTS: Here, we report the isolation of endophytic bacteria from root, stem, leaf and seed of three winter wheat varieties at tillering, jointing, heading and seed-filling growth stages that were identified via 16S rRNA gene sequence analysis. Strains were screened for indole-3-acetic acid (IAA) production, potassium and phosphate solubilisation and the ability to grow on a nitrogen-free medium. Strain's capacity to stimulate various plant growth parameters, such as dry root weight, dry above-ground parts weight and plant height, was evaluated in pot trials. A total of 127 strains were randomly selected from 610 isolated endophytic bacterial cultures, representing ten genera and 22 taxa. Some taxa were organ-specific; others were growth-stage-specific. Bacillus aryabhattai, B. stratosphericus, Leclercia adecarboxylata and Pseudomonas oryzihabitans were detected as wheat endophytes for the first time. The IAA production, inorganic phosphorous solubilisation, organic phosphorus solubilisation, potassium solubilisation and growth on N-free medium were detected in 45%, 29%, 37%, 2.4% and 37.8% of the 127 strains, respectively. In pot trials, each strain showed variable effects on inoculated wheat plants regarding the evaluated growth parameters. CONCLUSIONS: Wheat endophytic bacteria showed organ- and growth-stage diversity, which may reflect their adaptations to different plant tissues and seasonal variations, and differed in their PGP abilities. Bacillus was the most predominant bacterial taxa isolated from winter wheat plants. Our study confirmed wheat root as the best reservoir for screening endophytic bacteria with potential as biofertilisers.

RNA Interference-Mediated Knockdown of Bombyx mori Haemocyte-Specific Cathepsin L (Cat L)-Like Cysteine Protease Gene Increases Bacillus thuringiensis kurstaki Toxicity and Reproduction in Insect Cadavers.

The silkworm's Cat L-like gene, which encodes a lysosomal cathepsin L-like cysteine protease, is thought to be part of the insect's innate immunity via an as-yet-undetermined mechanism. Assuming that the primary function of Cat L-like is microbial degradation in mature phagosomes, we hypothesise that the suppression of the Cat L-like gene expression would increase Bacillus thuringiensis (Bt) bacteraemia and toxicity in knockdown insects. Here, we performed a functional analysis of Cat L-like in larvae that were fed mulberry leaves contaminated with a commercial biopesticide formulation based on Bt kurstaki (Btk) (i.e., Dipel) to investigate its role in insect defence against a known entomopathogen. Exposure to sublethal doses of Dipel resulted in overexpression of the Cat L-like gene in insect haemolymph 24 and 48 h after exposure. RNA interference (RNAi)-mediated suppression of Cat L-like expression significantly increased the toxicity of Dipel to exposed larvae. Moreover, Btk replication was higher in RNAi insects, suggesting that Cat L-like cathepsin may be involved in a bacterial killing mechanism of haemocytes. Finally, our results confirm that Cat L-like protease is part of the antimicrobial defence of insects and suggest that it could be used as a target to increase the insecticidal efficacy of Bt-based biopesticides.

Tandem Mass Tag-Based Quantitative Proteomics and Virulence Phenotype of Hemolymph-Treated Bacillus thuringiensis kurstaki Cells Reveal New Insights on Bacterial Pathogenesis in Insects.

The spore-forming bacterium Bacillus thuringiensis (Bt) of the Bacillus cereus group uses toxin-opened breaches at the insect midgut epithelium to infest the hemolymph, where it can rapidly propagate despite antimicrobial host defenses and induce host death by acute septicemia. The response of Bt to host hemolymph and the latter's role in bacterial pathogenesis is an area that needs clarification. Here, we report a proteomic analysis of the Bt kurstaki strain HD73 (Btk) hemolymph stimulon showing significant changes in 60 (34 up- and 26 downregulated) differentially accumulated proteins (DAPs). Gene ontology (GO) enrichment analysis revealed that DAPs were mainly related to glutamate metabolism, transketolase activity, and ATP-dependent transmembrane transport. KEGG analysis disclosed that DAPs were highly enriched in the biosynthesis of bacterial secondary metabolites, ansamycins. Interestingly, about 30% of all DAPs were in silico predicted as putative virulence factors. Further characterization of hemolymph effects on Btk showed enhanced autoaggregation in liquid cultures and biofilm formation in microtiter polystyrene plates. Hemolymph-exposed Btk cells were less immunogenic in mice, suggesting epitope masking of selected surface proteins. Bioassays with intrahemocoelically infected Bombyx mori larvae showed that hemolymph preexposure significantly increased Btk toxicity and reproduction within the insect (spore count per cadaver) at low inoculum doses, possibly due to 'virulence priming'. Collectively, our findings suggest that the Btk hemolymph stimulon could be partially responsible for bacterial survival and propagation within the hemolymph of infected insects, contributing to its remarkable success as an entomopathogen. All mass spectrometry data are available via ProteomeXchange with identifier PXD021830. IMPORTANCE After ingestion by a susceptible insect and damaging its midgut epithelium, the bacterium Bacillus thuringiensis (Bt) reaches the insect blood (hemolymph), where it propagates despite the host's antimicrobial defenses and induces insect death by acute septicemia. Although the hemolymph stage of the Bt toxic pathway is determinant for the infested insects' fate, the response of Bt to hemolymph and the latter's role in bacterial pathogenesis has been poorly explored. In this study, we identified the bacterial proteins differentially expressed by Bt after hemolymph exposure. We found that about 30% of hemolymph-regulated Bt proteins were potential virulence factors, including manganese superoxide dismutase, a described inhibitor of hemocyte respiratory burst. Additionally, contact with hemolymph enhanced Bt virulence phenotypes, such as cell aggregation and biofilm formation, altered bacterial immunogenicity, and increased Bt toxicity to intrahemocoelically injected insects.

Potato virus X-mediated constitutive expression of Plutella xylostella PxSDF2L1 gene in Nicotiana benthamiana confers resistance to Phytophthora parasitica var. nicotianae.

BACKGROUND: The Plutella xylostella PxSDF2L1 gene was previously reported to enhance insect resistance to pathogen at high basal transcription rate. PxSDF2L1 shows similitude with the stromal cell-derived factor 2 (SDF2), an ER stress-induced chaperon protein that is highly conserved throughout animals and plants. The precise biological function of SDF2 is not clear, but its expression is required for innate immunity in plants. Here, we investigate whether a continuous expression of PxSDF2L1 in Nicotiana benthamiana can similarly confer resistance to plant pathogen, particularly, the black shank Phytophthora parasitica var. nicotianae. RESULTS: The N. benthamiana plants were inoculated with agrobacteria transformed with a PVX-based binary vector carrying the PxSDF2L1 gene; similar agroinoculation experiments with a PVX vector carrying the GFP gene were used for controls. In pot trials, agroinfected N. benthamiana plants constitutively expressing PxSDF2L1 showed a significant reduction of stem disease symptoms caused by the inoculation with P. parasitica, compared with controls. CONCLUSIONS: We confirm a role of PxSDF2L1 in resistance to black shank, with a potential application to engineering active resistance against this oomycete in the commercial N. tabacum species and propose its evaluation in other crop families and plant pathogens.

First Report of Soft Rot Caused by Aspergillus niger sensu lato on Mother-in-law's Tongue in China.

"Mother-in-law's tongue" (MLT) [Dracaena trifasciata (Prain) Mabb. (syn. Sansevieria trifasciata Prain.)], also known as "Saint George's sword", "snake plant", "tiger's tail orchid", etc., is an evergreen perennial ornamental plant grown worldwide. In September 2016, severe soft rot occurred on the leaves of MLT in a flower market in Nanyang city (32º56´N, 112º32´E), Henan province, China with 25% disease incidence (n=100). Water-soaked spots initially appeared on the leaf margin, enlarged rapidly, and became soft rot under excessively watered conditions. A blight zone was visualized at the margin of a developing lesion in backlit conditions. Severely affected leaves folded down from the lesions. Lesion expansion stopped under dry conditions. Grey or dark brown mycelia were frequently seen on the lesions. Tissue pieces (4×4 mm2) at the margin of lesions were cut out, treated with 75% ethanol for 10 s, followed by 70 s in 0.1% HgCl2, rinsed eight times with sterile water, and plated on potato dextrose agar (PDA) medium. Pure Aspergillus cultures were obtained from the surface-disinfected lesions after 4 days of incubation at 26°C. Two single-spore-derived isolates (An-1 and An-2) were randomly selected and used for morphological and molecular identifications as well as pathogenicity tests. The isolates formed round dark brown colonies with a large number of conidia after 5 days of incubation on PDA at 28°C. Conidia were subsphaeroidal or oblate, unicellular, dark brown, 2.9-4.2(3.5) × 1.9-3.4(2.7) µm in size (n=100), developed from a two-series of strigmata born on a conidial head, with ridge or stab-shaped prominences. For pathogenicity tests, the two isolates were separately grown on oatmeal agar and incubated at 30°C for 6 days. Mycelial plugs (5 mm diam.) were inoculated on the scalpel incision X-shaped wounds of surface-disinfected leaves of MLT. The inoculated leaves were kept on a two-layer of wet napkin in a steel basin covered with a plastic film. Soft rot symptoms developed from the wounds 6 days after incubation, similar to those observed on naturally affected leaves. The An-1- and An-2-inoculated unwounded leaves remained symptomless during the pathogenicity tests. Fungal cultures with the same phenotypes as the inocula were consistently reisolated from the lesions of the leaves inoculated by each of the two isolates, verifying the isolates as the causal agent of the disease based on Koch's postulates. Both ß-tubulin gene and rDNA-ITS (internal transcribed spacer) sequences of the two isolates were separately amplified and sequenced. Sequences were submitted to GenBank with accession numbers MN259522 and MN259523 for the ß-tubulin gene sequences, and accession numbers MN227322 and MN227324 for the rDNA-ITS sequences of An-1 and An-2, respectively. Both An-1 and An-2 were clustered with members of Aspergillus niger van Tieghem in the phylogenetic tree of rDNA-ITS, clearly separated from other Aspergillus spp. In the phylogenetic tree of ß-tublin gene, both An-1 and An-2 formed a subclade inside a large clade consisting of members of A. niger in strict sense. Based on the molecular and morphological results, both An-1 and An-2 clearly separated from other Aspergillus spp. and can be considered as A. niger sensu lato. Foliar diseases of MLT are known to be caused by a few fungal species such as Chaetomella spp. (Li et al. 2014) and Colletotrichum sansevieriae (Nakamura et al. 2006). This is the first report of A. niger sensu lato causing soft rot on MLT in China.

Functional expression of a peritrophin A-like SfPER protein is required for larval development in Spodoptera frugiperda (Lepidoptera: Noctuidae).

Peritrophins are associated with structural and functional integrity of peritrophic membranes (PM), structures composed of chitin and proteins. PM lines the insect midgut and has roles in digestion and protection from toxins. We report the full-length cDNA cloning, molecular characterization and functional analysis of SfPER, a novel PM peritrophin A protein, in Spodoptera frugiperda. The predicted amino acid sequence indicated SfPER's domain structure as a CMCMC-type, consisting of a signal peptide and three chitin-binding (C) domains with two intervening mucin-like (M) domains. Phylogenetic analysis determined a close relationship between SfPER and another S. frugiperda PM peritrophin partial sequence. SfPER transcripts were found in larvae and adults but were absent from eggs and pupae. Chitin affinity studies with a recombinant SfPER-C1 peritrophin A-type domain fused to SUMO/His-tag confirmed that SfPER binds to chitin. Western blots of S. frugiperda larval proteins detected different sized variants of SfPER along the PM, with larger variants found towards the posterior PM. In vivo suppression of SfPER expression did not affect susceptibility of larvae to Bacillus thuringiensis toxin, but significantly decreased pupal weight and adult emergence, possibly due to PM structural alterations impairing digestion. Our results suggest SfPER could be a novel target for insect control.

Biocontrol Agents for Controlling Wheat Rust.

Bacterial endophytes are potential biocontrol agents of wheat rusts. Apart from disease control, these bacterial endophytes have growth-promoting efficacies which differ significantly from one isolate to another. Here, we describe the procedure for isolation, screening, and identification of endophytic bacterial isolates with high capacities to suppress strip rust infection and better ability to enhance wheat yields.

RNAi-mediated plant protection against aphids.

Aphids (Aphididae) are major agricultural pests that cause significant yield losses of crop plants each year by inflicting damage both through the direct effects of feeding and by vectoring harmful plant viruses. Expression of double-stranded RNA (dsRNA) directed against suitable insect target genes in transgenic plants has been shown to give protection against pests through plant-mediated RNA interference (RNAi). Thus, as a potential alternative and effective strategy for insect pest management in agricultural practice, plant-mediated RNAi for aphid control has received close attention in recent years. In this review, the mechanism of RNAi in insects and the so far explored effective RNAi target genes in aphids, their potential applications in the development of transgenic plants for aphid control and the major challenges in this regard are reviewed, and the future prospects of using plant-mediated RNAi for aphid control are discussed. This review is intended to be a helpful insight into the generation of aphid-resistant plants through plant-mediated RNAi strategy. © 2016 Society of Chemical Industry.

Characterization of a new pathovar of Agrobacterium vitis causing banana leaf blight in China.

A new banana leaf blight was found in Nanning city, China, during a 7-year survey (2003-2009) of the bacterial diseases on banana plants. Eight bacterial strains were isolated from affected banana leaves, and identified as an intraspecific taxon of Agrobacterium vitis based on their 16S rDNA sequence similarities with those of 37 randomly selected bacterial strains registered in GenBank database. The representative strain Ag-1 was virulent on banana leaves and shared similar growth and biochemical reactions with the reference strain IAM14140 of A. vitis. The strains causing banana leaf blight were denominated as A. vitis pv. musae. The traditional A. vitis strains virulent to grapevines were proposed to be revised as A. vitis pv. vitis. This is the first record of a new type of A. vitis causing banana leaf blight in China.

Engineering plants for aphid resistance: current status and future perspectives.

KEY MESSAGE: The current status of development of transgenic plants for improved aphid resistance, and the pros and cons of different strategies are reviewed and future perspectives are proposed. Aphids are major agricultural pests that cause significant yield losses of crop plants each year. Excessive dependence on insecticides for aphid control is undesirable because of the development of insecticide resistance, the potential negative effects on non-target organisms and environmental pollution. Transgenic plants engineered for resistance to aphids via a non-toxic mode of action could be an efficient alternative strategy. In this review, the distribution of major aphid species and their damages on crop plants, the so far isolated aphid-resistance genes and their applications in developments of transgenic plants for improved aphid resistance, and the pros and cons of these strategies are reviewed and future perspectives are proposed. Although the transgenic plants developed through expressing aphid-resistant genes, manipulating plant secondary metabolism and plant-mediated RNAi strategy have been demonstrated to confer improved aphid resistance to some degree. So far, no aphid-resistant transgenic crop plants have ever been commercialized. This commentary is intended to be a helpful insight into the generation and future commercialization of aphid-resistant transgenic crops in a global context.

Colonization of Caenorhabditis elegans by Bacillus nematocida B16, a bacterial opportunistic pathogen.

BACKGROUND: Soil-dwelling Bacillus nematocida B16 can kill Caenorhabditis elegans via a Trojan horse-like mechanism. However, colonization is a key problem that must be solved during the infection process. AIMS: To study the molecular mechanism involved in the colonization of B. nematocida B16 against the host C. elegans. METHODS: GFP-expressing strain B16g was constructed and its nematocidal activity was assayed. 'Feeding transfer' experiments were carried out separately using of B16 and B16g strains to explore the colonization mode of the bacteria. Fluorescence microscopy was used to observe the interactions between fluorescent signal and the quantity of bacteria in the intestine. A mariner-based transposon called TnYLB-1 was also applied in the random mutagenesis of B16 to screen the mutants with impaired colonization of nematode worms and identify potential localization-related genes. RESULTS AND CONCLUSION: A small inoculum of the bacteria resulted in its proliferation in the C. elegans intestine. The fluorescence signal was enhanced with increasing bacterial density in the intestine. Several candidate genes with possibly important roles in colonization were found. These results provide a solid foundation for further elucidation of the infection process at the molecular level and enrichment of our knowledge of bacterial pathogenesis.

[Field resistance of Phytophthora melonis to metalaxyl in South China].

OBJECTIVE: Phytophthora melonis is the casual agent of wax gourd and cucumber Phytophthora blight which becomes a constraint for sustainable production of the related crops. Metalaxyl is one of the principal fungicides for controlling the disease now. The objectives of the present study were: (1) to investigate the baseline sensitivity and field resistance of P. melonis to metalaxyl in South China; (2) to test the occurrence of metalaxyl-resistant mutants from metalaxyl-sensitive wild type strains exposed to the fungicide; and (3) to monitor the development of metalaxyl resistance in P. melonis population. METHODS: Over 400 samples of wax gourd and cucumber Phytophthora blight were collected from Guangxi Zhuang Autonomous Region and Guangdong province during 2007-2010, and 193 strains of P. melonis were isolated and purified. The sensitivity of the isolated strains to metalaxyl was tested using mycelial growth rate method in vitro and floating-leaf-disk method in vivo, respectively. The metalaxyl-sensitive strains were induced on PDA plates containing 10 microg/mL metalaxyl. RESULTS: The sensitive, moderately resistant and resistant strains were recorded as 29.0% , 18.1% and 52.8%, respectively, among 193 tested strains. The frequency and level of resistance of P. melonis from Guangdong were higher than that from Guangxi. The strains from cucumber was generally more resistant to metalaxyl than those from wax gourd. The metalaxyl-resistant strains were frequently detected as predominant populations in most of the sampling sites and the highest resistance index (4226.9) was confirmed. Metalaxyl-resistant (M1r) mutants could be isolated from approximately 60% of the sensitive wild-type strains. The resistance level of the M mutants was 189-407 times higher than that of their sensitive parental strains. The EC50 values of 9 sensitive strains from a sampling site without a record of phenylamide fungicide application ranged from 0.0429 to 0.5461 microg/mL. Their mean EC50 value (0.3200 +/- 0.1617 microg/mL) was considered as the baseline sensitivity of P. melonis to metalaxyl in South China. CONCLUSION: Metalaxyl-resistant strains universally occur in South China, especially in the vegetable-growing areas with a longer history of metalaxyl application. The establishment of the baseline sensitivity of P. melonis to metalaxyl will provide a science-based guide for evaluating and further monitoring resistance of the pathogen to the fungicide.

[Characterization of a bacterial biocontrol strain 1404 and its efficacy in controlling postharvest citrus anthracnose].

OBJECTIVE: Anthracnose caused by Colletotrichum gloeosporioides (Penz.) Sacc. is a main disease in citrus production. To develop an effective biocontrol measure against citrus postharvest anthracnose, we screened antagonistic microbes and obtained a bacterial strain 1404 from the rhizospheric soil of chili plants in Nanning city, Guangxi, China. The objectives of the present study were to: (1) identify and characterize the antagonistic bacterium; and (2) to evaluate the efficacy of the antagonistic strain in controlling citrus postharvest anthracnose disease. METHODS: Strain 1404 was identified by comparing its 16S rDNA sequence with related bacteria from GenBank database, as well as analyzing its morphological, physiological and biochemical characters. The antagonistic stability of the strain 1404 was determined by continuously transferring it on artificial media. The effect of the strain on suppressing citrus anthracnose at postharvest stage was tested by stab inoculation method. RESULTS: The 16S rDNA of strain 1404 was amplified with primers PF1 (5'-AGAGTTTGATCATGGCTCAG-3') and PR1 (5'-TACGGTTACCTTGTTACGACTT-3') and its sequence submitted to GenBank (accession number: GU361113). Strain 1404 clustered with the GenBank-derived Brevibacillus brevis strains in the 16S-rDNA-sequence-based phylogenetic tree at 100% bootstrap level. The morphological traits, physiological and biochemical characters of strain 1404 agreed with that of Brevibacillus brevis. Less change in the suppressive ability of antagonist against growth of Colletotrichum gloeosporioides was observed during four continuous transfers on artificial media. The average control efficacy of the strain was 64. 9 % against the disease 20 days after the antagonist application. CONCLUSION: Strain 1404 was identified as Brevibacillus brevis based on its morphological traits, phyiological and biochemical characters as well as 16S rDNA sequence analysis. The antagonist was approved to be a promising biocontrol agent. This is the first report of Brevibacillus brevis as an effective antagonist against citrus postharvest anthracnose disease.