Huifang Shen

Analysis of the defence-related mechanism in cucumber seedlings in relation to root colonization by nonpathogenic Fusarium oxysporum CS-20

A defence response can be induced by nonpathogenic Fusarium oxysporum CS-20 in several crops, but the molecular mechanism has not been clearly demonstrated. In the present study, we analysed the defence mechanism of a susceptible cucumber cultivar (Cucumis sativus L. 9930) against a pathogen (F. oxysporum f. sp. cucumerinum) through the root precolonization of CS-20. A challenge inoculation assay indicated that the disease severity index (DSI) was reduced, ranging from 18.83 to 61.67 in comparison with the pathogen control. Root colonization analysis indicated that CS-20 clearly did not appear to influence the growth of cucumber seedlings. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) revealed that CS-20-mediated defence response was activated by PR3, LOX1 and PAL1 and the pathogen-mediated resistance response was regulated by PR1 and PR3. Moreover, both nonpathogenic and pathogenic F. oxysporum were able to upregulate NPR1 expression. In contrast to a pathogen, CS-20 can activate the Ca(2+) /CaM signal transduction pathway, and the gene expression of both CsCam7 and CsCam12 increased significantly. The gene expression analysis indicated that CS-20 strongly enhanced the expression of PR3, LOX1, PAL1, NPR1, CsCam7 and CsCam12 after inoculation. Overall, the defence response induced by CS-20 can be controlled by multiple genes in the cucumber plant.

FocVel1 influences asexual production, filamentous growth, biofilm formation, and virulence in Fusarium oxysporum f. sp. cucumerinum.

Velvet genes play critical roles in the regulation of diverse cellular processes. In current study, we identified the gene FocVel1, a homolog of Fusarium graminearum VelA, in the plant pathogenic fungus F. oxysporum f. sp. cucumerinum. This pathogen causes the destructive disease called cucumber Fusarium wilt (CFW), which severely affects the production and marketing of this vegetable worldwide. Transcript analyses revealed high expression of FocVel1 during conidiophore development. Disruption of the FocVel1 gene led to several phenotypic defects, including reduction in aerial hyphal formation and conidial production. The deletion mutant ΔFocVel1 showed increased resistance to both osmotic stress and cell wall-damaging agents, but increased sensitivity to iprodione and prochloraz fungicides, which may be related to changes in cell wall components. In the process of biofilm formation in vitro, the mutant strain ΔFocVel1 displayed not only a reduction in spore aggregation but also a delay in conidial germination on the polystyrene surface, which may result in defects in biofilm formation. Moreover, pathogenicity assays showed that the mutant ΔFocVel1 exhibited impaired virulence in cucumber seedlings. And the genetic complementation of the mutant with the wild-type FocVel1 gene restored all the defects of the ΔFocVel1. Taken together, the results of this study indicated that FocVel1 played a critical role in the regulation of various cellular processes and pathogenicity in F. oxysporum f. sp. cucumerinum.

Identification of Dickeya zeae as a Causal Agent of Bacterial Soft Rot in Banana in China

Bacterial soft rot of banana was first noticed in 2009 in Guangzhou city, China. The disease also was observed on various banana cultivars of different genotypes in several other cities. Symptoms of the disease included leaf wilting, collapse of pseudostems, and unusual odor. Five isolated strains that fulfilled Kochs postulates were used for biochemical testing. The five strains were most similar to Dickeya dadantii or D. zeae, but were much less similar to D. paradisiaca when using several phenotype characteristics. Sequence analysis of 16S rDNA, dnaX, gryB, and recA of a reference strain revealed a similarity of 99% with the sequences of D. zeae, rather than D. paradisiaca. Phylogenic analysis of concatenated sequences of dnaX, gryB, and recA indicated that the banana strain constituted a distinguishable clade with several D. zeae strains involving rice pathogens D. zeae EC1 and ZJU1202 from Guangdong province, but the banana pathogen had several characteristics that distinguished it from the rice pathogens. Therefore, the banana pathogen was determined to be D. zeae. This is the first report of banana soft rot caused by D. zeae in China; however, the pathogen can infect other important crops.

Species-specific detection of Dickeya sp. ( Pectobacterium chrysanthemi ) in infected banana tissues, soil and water

Dickeya sp. ( Pectobacterium chrysanthemi ) is a bacterial pathogen that can cause soft rot disease on banana pseudostem tissue. A species-specific polymerase chain reaction (PCR) assay was developed for rapid and sensitive detection of the pathogenic bacteria in diseased plant tissues, soil and artificially infested irrigation water. Based on differences in internal transcribed spacer (ITS) sequences of Dickeya sp. ( P. chrysanthemi ) and other bacteria, a pair of species-specific primers was synthesized. Specificity was tested against eight bacterial organisms associated with banana. The LF/LR primers amplified only a single PCR band of 171 bp from Dickeya sp. (P. chrysanthemi ). The detection sensitivity was determined to be 0.44 fg for pure genomic DNA per 25 μl reaction volume. The results suggest that the assay detected the pathogen more rapidly and accurately than standard isolation methods. The PCR-based methods developed here could simplify both plant disease diagnosis and pathogen monitoring, as well as guide plant disease management. Key words: Soft rot, banana, detection, polymerase chain reaction (PCR).

First Report of Rust of Plumeria rubra Caused by Coleosporium plumeriae in Guangdong Province, China

Plumeria spp. are ornamental trees commonly planted in parks and gardens, and Plumeria rubra cultivars (Frangipani) is most common in Guangdong Province, China. A rust disease of P. rubra was observed on leaves of susceptible plants from August to December 2013. Ten nurseries were surveyed in September 2013, and 91% (220 of 240) of the plumeria plants were infected with rust. Symptoms first appeared as chlorotic spots (about 1 mm in diameter) appearing on adaxial leaf surfaces and then spread to whole leaf, and infection further resulted in leaf necrosis and abscission. Therefore, the ornamental value of diseased trees was greatly diminished. Bright yellow or yellow-orange uredinia were hypophyllous and produced under the epidermis. Urediniospores were catenulate, globose, ovoid or ellipsoid, and sometimes angular in appearance, ranging from 20.0 to 42.0 μm in length by 14.1 to 25.6 μm in width. Their walls were verrucose and 1.3 to 3.2 μm thick. No teliospores were observed. The rust was identified as Coleosporium plumeriae Pat. based on urediniospore morphology (2). Species identity was confirmed with a 1,551-bp sequence (GenBank Accession No. KF879087) of ITS rDNA amplified with rust-specific primers Rust2inv and LR6 (1). The amplicon had a 100% similarity to C. plumeriae (GU145555). Pathogenicity was confirmed by spraying a urediniospores suspension (15,000 spores ml-1) on five plants of P. rubra cultivar. Five leaves of each plant were inoculated and sealed in plastic bags, while five control plants were applied with sterile water. Plants were held at 28°C for 36 h in a dew chamber. All inoculated leaves developed typical rust symptoms with the uredinia appearing after 9 days, no symptoms developed on any control plants. Urediniospores were produced on infected leaves and pathogen identity was confirmed by morphology and re-sequencing of the ITS rDNA. Plumeria rust was first found in Hong Kong (4) and then in Hainan and Yunnan Provinces, China (3). However, this is the first report of plumeria rust in Guangdong Province, China. Frangipani has large, colorful flowers in the summer, and this rapidly spreading disease causes severe damage and affects their aesthetic value in the second half of the year. References: (1) M. C. Aime. Mycoscience 47:112, 2006. (2) N. Patouillard. Bull. Soc. Mycol. Fr. 18:171, 1902. (3) Q. Wang et al. New Dis. Rep. 23, doi:10.5197/j.2044-0588.2011.023.010, 2011. (4) J. Yan et al. Mycosystema 25:327, 2006 (in Chinese).

Genome Sequence of the Banana Pathogen Dickeya zeae Strain MS1, Which Causes Bacterial Soft Rot.

ABSTRACT We report a draft genome sequence of Dickeya zeae strain MS1, which is the causative agent of banana soft rot in China, and we show several of its specific properties compared with those of other D. zeae strains. Genome sequencing provides a tool for understanding the genomic determination of the pathogenicity and phylogeny placement of this pathogen.

Characterization of Canna yellow mottle virus in a New Host, Alpinia purpurata, in Hawaii

Canna yellow mottle virus (CaYMV) is an important badnavirus infecting Canna spp. worldwide. This is the first report of CaYMV in flowering ginger (Alpinia purpurata) in Hawaii, where it is associated with yellow mottling and necrosis of leaves, vein streaking, and stunted plants. We have sequenced CaYMV in A. purpurata (CaYMV-Ap) using a combination of next-generation sequencing and traditional Sanger sequencing techniques. The complete genome of CaYMV-Ap was 7,120 bp with an organization typical of other Badnavirus species. Our results indicated that CaYMV-Ap was present in the episomal form in infected flowering ginger. We determined that this virus disease is prevalent in Hawaii and could potentially have significant economic impact on the marketing of A. purpurata as cut flowers. There is a potential concern that the host range of CaYMV-Ap may expand to include other important tropical plants.

First Report of Pineapple Heart Rot Caused by Phytophthora nicotianae in Hainan Province, China

Pineapple (Ananas comosus) is an economically important tropical fruit in Hainan Province, China. During September to November 2011, heart rot disese of pineapple was found in Ledong and Wangning of Hainan Province. A survey of 150 ha producing areas of pineapple revealed that the fields were affected at an incidence ranging from 25% to 30%. Infected plants showed water-soaked lesions and soft rot on the base of heart leaves near the soil surface. Heart leaves of infected plants were easily pulled out. As the disease progressed, plants collapsed and died. Diseased tissue fragments (2 × 2 mm) were surface-disinfected for 10 min with 0.3% NaClO, then rinsed three times in sterile water, and plated to 10% V8 juice agar (4). Inoculated dishes were incubated at 26°C in the dark. After 5 days, Phytophthora (identified by the presence of coenocytic hyphae and papillate sporangia) were isolated from the tissue cultures, which has aseptate hyphae. Sporangia were papillate, noncaducous, oval or spherical, and 34.5 to 58.2 μm. Clamydospores, both terminal and intercalary, were also spherical, and were 23.4 to 34.0 μm (2). The ITS region of rDNA was amplified using primers ITS4/ITS5, and the 927-bp product of the ITS showed 99% sequence identity to Phytophthora nicotianae (GenBank Accession No. JF792540), and the sequence was accessed to NCBI (JX978446). Pathogenicity tests were confirmed by irrigating the wounded stem bases of 10 2-month-old pineapple plants with 50 ml of P. nicotionae zoospore solution (15,000 zoospores/ml), and another 10 plants of the same cultivar inoculated with sterile water served as controls. Plants were placed in pots in a greenhouse at 28°C and 90% relative humidity. After 9 days, soft rot was observed clearly on the base of heart leaves of all 10 inoculated plants, while the control plants appeared normal. P. nicotianae was reisolated from the infected pineapple plants, and confirmed to be the same as the inoculated pathogen by conducting a ITS rDNA sequence comparison and morphological characteristics. P. nicotianae was previously reported as the causal agent of heart rot of pineapple in Hawaii, U.S.A. (3) and Guangdong Province of China (1). To our knowledge, this is the first report of P. nicotianae on pineapple in Hainan Province, China. References: (1) J. Z. Chen et al. J. Yunnan Agric. Univ. 8:134, 2003. (2) H. H. Ho. Mycologia 73:705, 1981.(3) K. W. Howard et al. Plant Dis. Rep. 48:848, 1964. (4) X. B. Zheng. Page 81 in: Phytophthora and its Research Technology. China Agricultural Press, Beijing, 1997.

First Report of Bacterial Soft Rot on Tagetes patula Caused by Dickeya dieffenbachiae in China

French marigold (Tagetes patula L.), originally from Mexico, is an annual herb widely planted in China because of its beautiful color, long flowering, and strong adaptability, and has been used widely for ornamentation and decorating. French marigold is also rich in patuletin, quercetagetin, and patulitrin, and is therefore applied medicinally for treating colds and coughs. In early summer 2012, soft rot symptoms on French marigold were found at three flower nurseries in Guangzhou, Guangdong Province, P. R. China, and approximately 25% of the plants had the symptoms. The symptoms included tissue collapse of the stems at the soil line followed by wilting of the whole plants. Within 1 week, the infected stems showed vascular discoloration, turned brown and then inky black, and eventually the whole plant collapsed after the basal stem was infected. Bacteria were successfully isolated from eight symptomatic plants on nutrient agar media incubated at 30°C for 48 h. Ten isolates were selected randomly for further characterization. They were gram negative, degraded pectate, negative for oxidase and positive for indole production, and utilized malonate, glucose, and sucrose but not glucopyranoside, trehalose, or palatinose. Polymerase chain reactions (PCR) were performed using the 16S primers 27f and 1495r (4) for molecular identification. Subsequent DNA sequencing showed that the representative tested strain TP1 (GenBank Accession No. JX575747) was 99% identical to that of Dickeya dieffenbachiae (JF419463) using BLASTn. Further genetic analysis of strain TP1 was performed targeting several housekeeping genes, i.e., dnaX (GenBank Accession No. JX575748) with primers dnaxf and dnaxr (3), gyrB (JX575749) with primers of gyrbf1 and gyrbr1 (1), and gapA (JX575750) with primers of gapa326f and gapa845r (2). They were most homologous to the sequences of D. dieffenbachiae, since they had 97%, 96%, and 97% identity with GenBank accessions GQ904794, JF311653, and GQ891968, respectively. Pathogenicity was confirmed by injecting all 10 original bacterial isolates into each of 10 French marigold seedlings, with approximately 100 μl of a bacterial suspension at 1 × 108 CFU/ml. Ten plants inoculated with 100 μl of sterile water served as controls. Plants were placed in a greenhouse at 30 to 32°C and 90% relative humidity. Within 48 h, soft rot symptoms appeared on all inoculated seedlings, while the control plants appeared normal. D. dieffenbachiae was reisolated from the diseased tissues, and confirmed to be the same as the inoculated pathogen by conducting a 16S rDNA sequence comparison. Previously, black spot, botrytis blight, oedema, powdery mildew, southern bacterial wilt, and damping off have been found on T. patula. To our knowledge, it is the first report of a soft rot caused by D. dieffenbachiae on French marigold. Because of the popularity and high economic value of French marigold, identification of this progressing bacterial disease is important to maintain safe production and beautiful scenery. References: (1) B. R. Lin et al. Plant Dis. 96:452, 2012. (2) S. Nabhan et al. Plant Pathol. 61:498, 2012. (3) M. Sławiak et al. Eur. J. Plant Pathol. 125:245, 2009. (4) W. G. Weisburg. J. Bacteriol. 173:697, 1991.