Bao-Ju Li

Research status of clubroot (Plasmodiophora brassicae) on cruciferous crops in China

Abstract Clubroot, caused by Plasmodiophora brassicae, has emerged as a serious disease threatening the cruciferous crop production industry in China. The distribution, occurrence, physiological races and transmission routes of P. brassicae are briefly described here, providing a basis for understanding the serious implications of clubroot disease in China. Strategies for managing clubroot in a Chinese context are also discussed. Approximately 3.2–4.0 million ha of cruciferous crops are infected by P. brassicae every year in China, causing yield losses of 20–30%. The identification of physiological races is of fundamental importance for breeding clubroot-resistant cultivars. Race 4 of the pathogen, as classified on the differentials of Williams, has been spreading all over China and is becoming increasingly damaging to cruciferous crops. Dissemination of P. brassicae is mainly via the movement of resting spores on seed or in infected plant material. Spore dispersal over short distances depends on the movement of infested soil, wind, water and livestock manure. Integrated control strategies include the development of resistant cultivars, soil liming, fungicide applications and use of biological control agents.

First Report of Myrothecium Leaf Spot of Common Bean in China Caused by Myrothecium roridum

Common bean (Phaseolus vulgaris L.) is an economically important crop in China. In June 2008, a new foliar disease was observed on beans in Shunyi District, Beijing, China. The disease occurred in approximately 15% of the plants in a commercial field. Leaf spots were circular to irregular, reddish brown, zonate, and 8 to 20 mm in diameter. Black sporodochia with white tuffs were present on older lesions and black spore masses were present in moist weather. Ten isolates recovered from lesions produced white, floccose colonies and spore masses after 4 days on potato dextrose agar. The rod-shaped, hyaline conidia had rounded ends and averaged 6.8 × 2.5 μm. All characteristics were consistent with the description of Myrothecium roridum Tode ex Fr. (1). The internal transcribed spacer regions of one isolate were sequenced and deposited in GenBank (Accession No. GQ 381291). Sequences of the isolate from bean in China were 98% similar to sequences of M. roridum in GenBank. To determine pathogenicity, 30 healthy seedlings of common bean were inoculated by spraying a 1 × 105 conidia ml-1 suspension of M. roridum onto the foliage. Ten seedlings were sprayed with sterile water and served as controls. Plants were kept in a humid chamber at 27°C overnight and then placed in a growth chamber. After 6 days, the symptoms described above were observed on leaves in all inoculated plants, whereas symptoms did not develop on the control plants. The pathogen was reisolated from inoculated leaves, fulfilling Kochs postulates. There is one report of M. roridum on soybean in Korea (2). To our knowledge, this is the first report of Myrothecium leaf spot on common bean in China. References: (1) M. Fitton et al. CMI Mycol. Pap. No. 253, 1970. (2) K. J. Yum et al. Plant Pathol. J. 6:313, 1990.

Emergence of Bacterial Soft Rot in Cucumber Caused by Pectobacterium carotovorum subsp. brasiliense in China

During 2014 to 2015, a devastating bacterial soft rot on cucumber stems and leaves occurred in Shandong, Shanxi, Hebei, Henan, and Liaoning provinces of China, resulting in serious economic losses for cucumber production. The gummosis emerged on the surface of leaves, stems, petioles, and fruit of cucumber. The basal stem color was dark brown and the stem base turned to wet rot. Yellow spots and wet rot emerged at the edge of the infected cucumber leaves and gradually infected the leaf centers. In total, 45 bacterial strains were isolated from the infected tissues. On the basis of phenotypic properties of morphology, physiology, biochemistry, and 16S ribosomal RNA gene sequence analysis, the pathogen was identified as Pectobacterium carotovorum. Multilocus sequence analysis confirmed that the isolates were P. carotovorum subsp. brasiliense, and the pathogens fell in clade II. The pathogenicity of isolated bacteria strains was confirmed. The strains reisolated were the same as the original. The host range test confirmed that strains had a wide range of hosts. As far as we know, this is the first report of cucumber stem soft rot caused by P. carotovorum subsp. brasiliense in China as well as in the world, which has a significant economic impact on cucumber production.

Identification of Colletotrichum capsici (Syd.) Butler causing anthracnose on pumpkin in China

Abstract A severe disease was observed on pumpkin (Cucurbita moschata) in Beijing, China during 2009–2011. The causal agent was isolated from infected tissues, and its pathogenicity was confirmed. The pathogen was identified as Colletotrichum capsici (Syd.) Butler based on morphological characters and molecular methods. This is the first report of C. capsici causing anthracnose of pumpkin in China.

First report of crown rot of grafted cucumber caused by Fusarium solani in China.

Grafting has been widely and effectively used in cucumber (Cucumis sativus) cultivation for approximately 30 years in China to avoid Fusarium wilt caused by Fusarium oxysporum Schl. f. sp. cucumerinum Owen. In greenhouses, 90% of cucumbers are grafted onto pumpkin (Cucurbita moschata) rootstock. However, in March 2009, a severe crown rot causing yellowing and wilting of the leaves was observed on grafted cucumber in a large number of greenhouses in Lingyuan, western Liaoning Province in China. Symptoms consisted of dark brown, water-soaked lesions and a dense, white mycelial mat at the base of the stem. Lingyuan is the largest district for cucumber cultivation using grafting techniques in solar greenhouses in China. In 30 surveyed greenhouses in Sanshijiazi Village in the city of Lingyuan, the incidence of affected plants ranged from 10 to 40%, which caused serious economic losses. Fusarium spp. were isolated from the surface-sterilized basal stems of symptomatic plants on potato dextrose agar and incubated on potato sucrose agar for 4 days at 25°C. Colonies of the isolates produced a brown pigmentation and sparse, aerial mycelia, with a cream color on the underside. Conidiophores were elongated and branched or unbranched. Microconidia were abundant, hyaline, ellipsoid to ovoid, and 6 to 14 × 2.5 to 3.5 μm. Macroconidia were cylindrical, abundant, mostly two to six septate, 22 to 63 × 3.2 to 5.0 μm, with the apical cell rounded and blunt, and the basal cell rounded. On the basis of morphological characteristics, the fungus was identified as F. solani (C. Booth). For confirmation, the internal transcribed spacer region of rDNA was amplified and sequenced. A 449-bp sequence shared 99% homology with that of a F. solani GenBank accession previously reported from Japan (No. AF150473.1). The new sequence was deposited in GenBank (Accession No. HM015882). Pathogenicity of three isolates was determined in two experiments using different methods of inoculation. In one, 30 seedlings of pumpkin (C. moschata) with one true leaf each were inoculated by dipping their roots in a suspension of 106 spores ml-1, while control plants were mock inoculated with sterile water. Plants were then potted in a sterile mix of peat moss and vermiculite (2:1 vol/vol). In the other, pregerminated pumpkin seeds were sown in the same medium with a conidial suspension added at a rate of 106 spores ml-1, while other seeds were sown in sterile soil as controls. Plants for both experiments were maintained in a greenhouse at 25°C. Twelve days after inoculation, inoculated plants in both experiments showed a cortical rot on the crown and stem base with a brown, water-soaked appearance. Twenty-one days later, inoculated plants developed wilting and yellowed leaves. Disease incidence was 100%. No symptoms occurred on the control plants. Both experiments were repeated once with the same results. The pathogen was recovered from symptomatic tissue, confirming Kochs postulates. F. solani has been previously reported to cause root rot on cucurbit in California (2) and crown rot on grafted cucumber in the Netherlands (1). To our knowledge, this is the first report of crown rot of grafted cucumber caused by F. solani in China. References: (1) L. C. P. Kerling and L. Bravenboer. Neth. J. Plant Pathol. 73:15, 1967. (2) T. A. Tousson and W. C. Snyder. Phytopathology 51:17, 1961.

Evaluation of a loop-mediated isothermal amplification assay based on hrpZ gene for rapid detection and identification of Pseudomonas syringae pv. lachrymans in cucumber leaves.

Cucumber angular leaf spot caused by Pseudomonas syringae pv. lachrymans (Psl) is an important and destructive disease worldwide, and no effective technique has been developed for the control of the pathogen. Detection of infection or latent in cucumber plants is critical to evaluate disease progress and strengthening management to avoid a serious epidemic in the fields. In this paper, we developed a rapid and sensitive method for detection of Psl using an isothermal method known as loop‐mediated amplification (LAMP).

Boeremia leaf and fruit spot of okra caused by Boeremia exigua in China

Abstract Okra (Abelmoschus esculentus) is an economically important plant in China which is widely used in food, medicine and manufacturing. In September 2014, diseased samples of okra were collected in Yanqing District, Beijing City, China. Symptoms were brown to brownish black, sunken cavities on the leaves and fruits. The disease occurred on okra plants with an incidence of 35–55% in different fields. According to morphological and cultural characteristics, and sequence analysis of the internal transcribed spacer (ITS) and actin (ACT) regions, the pathogen was identified as Boeremia exigua (Desm.) Aveskamp, Gruyter & Verkley. Pathogenicity of the isolated fungus was conducted on okra fruits and on potted plants. Symptoms similar to those occurring in the field were reproduced. This is the first report of Boeremia spot of okra caused by Boeremia exigua in China.

Rapid detection and quantification of viable Pseudomonas syringae pv. lachrymans cells in contaminated cucumber seeds using propidium monoazide and a real-time PCR assay

Abstract Cucumber angular leaf spot, caused by Pseudomonas syringae pv. lachrymans (Psl), is one of the most devastating bacterial diseases in cucumber worldwide. Seedborne transmission of the pathogen is one of the principal modes for disease spread. To date, the detection of Psl is mainly achieved by culture methods, which are time and labour-consuming. In the present work, propidium monoazide (PMA) treatment combined with a quantitative real-time PCR (PMA-qPCR) assay was developed for quantifying viable Psl cells in contaminated cucumber seeds. PMA selectively penetrates compromised membranes of dead cells and inhibits DNA amplification during real-time PCR. The primers, based on a 162-bp amplicon from gap1 gene, were highly specific for Psl at the species level. PMA at 60 μmol L−1 was suitable for selective quantification of viable cells. The limit of detection (LOD) of PMA-qPCR for detecting viable cells in bacterial suspension and artificially contaminated cucumber seeds was 3.25 × 102 CFU·mL−1 and 47.73 CFU·g−1, respectively. For naturally contaminated seeds, quantifiable levels of viable cells were observed in eight out of the 37 samples, and ranged from 103 to 104 CFU·g−1. This assay has been confirmed to be a rapid and selective method to quantify the viable cells of Psl in bacterial suspension and cucumber seeds. Application of the assay may potentially improve pathogen control and disease management.

Grey mould caused by Botrytis cinerea on greenhouse-grown fennel in China

Abstract Leaf and stem lesions with symptoms of grey mould were observed on greenhouse-grown fennel (Foeniculum vulgare) plants in Beijing, China during 2013−2014. A fungus was isolated from lesions on leaves and stems. The pathogenicity of the isolated fungus was confirmed from inoculation experiments. On the basis of morphological features and ITS-5.8S rDNA, G3PDH, hsp60 and RPB2 sequences, the pathogen was identified as Botrytis cinerea Pers. This is the first report of grey mould caused by B. cinerea on fennel in China.

Corynespora spot of hot pepper caused by Corynespora cassiicola in China

Abstract Leaf and fruit spots and stem lesions were observed on hot pepper (Capsicum annuum L.) plants in Hainan province, China during 2010–2011. A fungus was isolated from dark brown spots on leaves and fruits and black stem lesions. The pathogenicity of the isolated fungus was confirmed from inoculation experiments. On the basis of morphological features and ITS-5.8S rDNA sequences, the pathogen was identified as Corynespora cassiicola (Berk. & Curt.) Wei. This is the first report of corynespora spot caused by C. cassiicola on hot pepper in China.