董偉清 何芳練 江文 高美萍 張尚文 王艷 韋紹龍 蔣慧萍 陳麗娟 閉志強(qiáng) 歐昆鵬 顏梅新

摘要：【目的】鑒定荔浦芋疫病病原及制備原生質(zhì)體，為荔浦芋病原檢測(cè)、致病機(jī)理研究及健康種苗生產(chǎn)提供科學(xué)依據(jù)?！痉椒ā繉?duì)采集自廣西荔浦縣芋頭種植區(qū)的芋疫病標(biāo)樣進(jìn)行分離，通過形態(tài)特征和rDNA-ITS分子生物學(xué)相結(jié)合的方法對(duì)其病原進(jìn)行鑒定，同時(shí)對(duì)獲得的病原菌進(jìn)行原生質(zhì)體制備?！窘Y(jié)果】通過對(duì)分離的芋疫病病原進(jìn)行致病性測(cè)定、形態(tài)特征觀察，將引起荔浦芋疫病的病原初步鑒定為芋疫霉菌（Phytophthora colocasiae）；rDNA-ITS序列分析結(jié)果表明，菌株DNA序列與GenBank已發(fā)表的P. colocasiae不同分離物序列同源性達(dá)99%，進(jìn)一步確定所測(cè)菌株為芋疫霉菌。制備獲得的芋疫霉菌原生質(zhì)體呈透明圓形或近圓形，大小不一。【結(jié)論】引起荔浦芋疫病的病原為芋疫霉菌，制備的芋疫霉菌原生質(zhì)體可用于芋疫霉病致病機(jī)理研究。

關(guān)鍵詞： 荔浦芋；疫病；芋疫霉菌；鑒定；原生質(zhì)體

Abstract：【Objective】The present study was conducted to identify the causal pathogen of taro leaf blight disease by using morphology and molecular methods， and establish a protoplast preparation protocol， in order to provide scientific basis for pathogen detection of taro leaf blight， pathogenesis and virus-free and healthy seedling production. 【Method】The samples were collected from taro production area in Lipu county， Guangxi. The pathogen was isolated， and identified through morphological examination combined with rDNA-ITS sequence analysis. Protoplast of pathogen was prepared by enzymatic digestion.【Result】The taro leaf blight pathogen was preliminarily identified as Phytophthora colocasiae based on its morphological characteristics and pathogenicity test. The results of rDNA-ITS sequence analysis indicated that the nucleotide sequence of the isolate was 99% similar to the isolates of P. colocasiae strains available in GenBank， which confirmed further that the isolate was P. colocasiae. Furthermore， the prepared protoplasts of P. colocasiae were circular or nearly circular， small or large， transparent.【Conclusion】The causal pathogen of taro leaf blight in Lipu of Guangxi is identified as P. colocasiae and its prepared protoplast could be used for pathogenesis study of taro leaf blight disease.

Key words： Colocasia esculenta（L.） Schott var. Lipu Taro； leaf blight； Phytophthora colocasiae； identification； protoplast

CLC number： S435.661 Document code： A Article：2095-1191（2016）11-1861-06

0 Introduction

【Research significance】Taro[Colocasia esculenta（L.） Schott]， a Colocasia member of the Araceae family， is originated in China（Yang and Liu， 1990； Wei and Ma， 1998） and India（Sharma et al.， 2008， 2009）. The tuber of taro could be used as staple food for millions of people in developing countries of Asia， Africa and central America（Misra et al.， 2008） because of rich source of proteins， carbohydrates， vitamins and minerals and its medicinal properties（Zhao et al.， 2002）. In China， cultivation area of taro is mainly located in Pearl River Valley area， especially in Guangxi being geographical advantages. Colocasia esculenta（L.） Schott var. Lipu Taro has been regarded as famous geographical indication products of Guangxi. Leaf blight of taro， caused by Phytophthora colocasiae， is one of the most destructive disease of taro（Zhang and Peng， 1994）， and lead to serious yield losses. With adjustment of agricultural industrial structure and expanding of the acreage of Lipu Taro， leaf blight of taro became more serious because of large planting area. Further more， it is difficult to study leaf blight pathogen of taro and its control at production at molecular level being unclear pathogenicity mechanism. Therefore， it is necessary to study the casual organism of leaf blight of Lipu Taro. 【Research progress】Leaf blight of taro was reported firstly in India in 1913（Butle and Kulkarni， 1913）， subsequently it spread to taro producing regions of east and north India（Misra， 1991）. Taro planting in Papua New Guinea were reported to have been devastated by the disease（Packard， 1975）. The disease has caused severely reduction in taro production in American Samoa（Gurr，1993）. Several observations of taro leaf blight at various regions of China were reported since 1980s（Wang，1995）. The causal pathogen of taro leaf blight was firstly identified as P. colocasiae in 1890 by Raciborski（Misraet al.，2008）. Subsequently， this pathogen was characterized in the aspects including morphological characters（Lu et al.， 2013； Ye et al.， 2016）， biological characteristics（Misra， 1996； Wang et al.， 2001； Ye et al.， 2016）， and disease management（Adams， 1999； Misra et al.， 2007）. With the application of rDNA-ITS analysis in identification of microbial species（Cooke and Duncan， 1997）， P. colocasiae isolates were collected from different states of India and subjected to ITS analysis to compare their sequence homology and construct the phylogenetic tree（Misra et al.，2011）. All isolates of P. colocasiae with higher identity located in a single cluster of the phylogenetic tree， regardless of their geographic origins（Misra et al.， 2011）. In China， all of the P. colocasiae isolates originated from southeastern Guangxi showed closely identical to the P. colocasiae isolates published on NCBI， with a homology of 97%-100%（Lu et al.， 2013）. Similar results were obtained by analyzing the ITS sequencing of P. colocasiae isolates collected from Guangdong， Guangxi， Hunan and Hubei in China（Zhou et al.， 2012）. 【Research breakthrough point】At present， the occurrence， pathogen morphological identification， and comprehensive controls of Lipu Taro leaf blight were conducted by many researchers； while the morphological characterization and molecular identification of causal pathogen of Lipu Taro leaf blight are still unreported， as well as protocol for protoplasts preparation of P. colocasiae. 【Solving problems】The identification of the causal organism of leaf blight of Lipu Taro by means of morphological characteristics and molecular biology methods were conducted， as well as establishment of protocol for protoplasts preparation of this pathogen， would provide scientific basis for pathogen detection and further investigation of its pathogenicity， and for healthy seedling production of taro in practice.

1 Materials and methods

1. 1 Experimental materials

The leaf blight sample of Lipu Taro was collected in taro cultivating area of Qingshan town in Lipu county of Guilin city in Guangxi on September 20， 2015.

1. 2 Experimental methods

1. 2. 1 Pathogen isolation Infected tissues with characteristic symptom from taro leaves were rinsed with 75% alcohol from 10 to 30 s， surface-sterilised in 3% sodium hypochlorite from 30 to 60 s and rinsed twice in distilled water（Fang， 1998）. Then the tissues were incubated on Phytophthora selective media of 10% V8 plates（Lu et al.， 2013）， which supplemented with 20 mg/L of rifamycin， 200 mg/L of ampicillin， 10 mg/L of pentachloronitrobenzene and carbendazol， and cultured at 28 ℃ for 3-4 days. Emerging mycelia were then transferred to 10% V8 medium and incubated for 4-5 days at 28 ℃. Isolated strains were stored in the incubator at 20 ℃.

1. 2. 2 Microscopy For microscopic analyses， isolated strains were cultured in 10% V8 medium for 4 days. Mycelia were then placed on the glass slide（Li et al.， 2007a）， observed and imaged using an Axi-

oimager Z1 microscope equipped with an AxiocamMRm camera（Carl Zeiss）.

1. 2. 3 Pathogenicity assay Pathogenicity test was conducted by method described as Zhou et al.（2012）， with some modification. Healthy taro leaves were placed on petri-dishes in diameter of 15 cm， with two layers of humid gauze. The 20 μL of zoospores suspension， at concentration of 106 spores/mL was inoculated on abaxial surfaces of young taro leaves， with ddH2O treatment as control（Misra et al.， 2011）. After inoculation， petri-dishes with leaves were covered with plastic bag and incubated at room temperature for 4 days. Re-isolation from resulting lesions was carried out according to Kochs postulates.

1. 2. 4 Molecular identification Total DNA isolation of P. colocasiae was conducted by method described as（Zhang et al.，2007）， with some modification. The rDNA internal transcribed spacer（ITS） region of isolates was amplified with primer pairs of ITS4（5'-TCC

TCCGCTTATTGATATGC-3'） and ITS5（5'-GGAAGT

AAAAGTCGTAACAAGG-3'）. The cycling conditions of PCR were as follows： initial denaturation of 94 ℃ for 2 min； 30 cycles of 94 ℃ for 30 s， 55 ℃ for 30 s， 72 ℃ for 1 min； and final elongation at 72 ℃ for 10 min. Amplified PCR product was analyzed by electrophoresis in a 1.0% of agarose gel. PCR-amplified fragment was sequenced by Invitrogen Trading Co.， Ltd（Shanghai）. The nucleotide sequence was blasted in NCBI（https：//blast.ncbi.nlm.nih.gov/Blast.cgi） and further analyzed. The identification of pathogen was determined finally based on the identity of the sequence and morphology of the isolate.

1. 2. 5 Protoplasts preparation of P. colocasiae Protoplasts were produced following a slightly modified protocol of McLeod et al.（2008）. The isolate of P. colocasiae was cultured on NPB medium plates at 25 ℃ in dark for 3-4 days. Several pieces of mycelium on agar（10 mm×10 mm） from the actively growing margin of colonies， were transferred to culture flask containing 100 mL of NPB liquid medium and were incubated in dark at 25 ℃ for 3 days. Mycelia of P. colocasiae was then harvested and rinsed twice with 0.8 mol/L Mannitol solution. The mycelia were subsequently added to 20 mL of enzyme digestion solution （lysing enzyme 0.15 g，cellulase 0.06 g，0.8 mol/L Mannitol 10 mL，ddH2O 8 mL，0.5 mol/L KCl 800 μL，0.5 mol/L MES 800 μL，0.5 mol/L CaCl2 400 μL） and were digested at room temperature with aggitation of 60 r/min for 45-60 min until abundant protoplast release occurred. The protoplasts were then washed in W5 Buffer and spun down by centrifuge at 1500 r/min at 4 ℃ for 3 min. The protoplasts were re-suspended in W5 Buffer to final concentration of 2×107 protoplasts/mL and placed on ice for 30 min. The protoplasts suspension in W5 Buffer were spun down again， at 1500 r/min at 4 ℃ for 3 min. After inoculation at room temperature for 10 min， the protoplasts were re-suspended in MMg solution.

2 Results and analysis

2. 1 Morphology characteristics

A strain was isolated from infected tissues with symptom characteristic of water-soaked spots and lesions（Fig.1-A）. Such isolated strain was allowed to grow on 10% V8 medium at 28 ℃ for 3-4 days. Colony of the isolate was cottony， round， light yellow to yellow-white（Fig.1-B）. Aerial hyphae were less， undivided， colorless and transparent， about 3.80-6.84 μm of breadth（Fig.1-C）. The isolate developed semipapillate and caducous sporangia on long pedicels， which were mostly ellipsoid to ovoid（Fig.1-D and Fig.1-E）. The results of the pathogenicity test revealed that all inoculated sites showed water-soaked lesions after four days inoculation of the isolate. Re-isolation from resulting lesions indicated that the isolates were identical to the inoculated one.

2. 2 ITS identification

The ITS region of isolate was successfully amplified with the primer pairs of ITS4/ITS5 and sequenced. An expected 890 bp product was yielded by PCR amplification. The DNA sequence（Fig.2） was compared to GenBank database using BLASTn for species identification. The ITS sequence analysis revealed that the nucleotide sequence of the isolate was 99% of similarity to the isolates of P. colocasiae available in GenBank. Ultimately， the causal organism of leaf blight of Lipu Taro was identified as P. colocasiae according to its pathogenicity， morphological characteristics and ITS DNA sequence analysis.

2. 3 Protoplasts preparation for P. colocasiae

Large numbers of protoplasts of P. colocasiae were generated successfully， after digestion of mycelia in enzyme solution for 50 min. Protoplasts of P. colocasiae were circular or nearly circular， small or large， transparent， when observed under the microscope（Fig.1-F）.

3 Discussion

Traditional identification of Phytophthora species is based on colony characteristics， sexual bodies， sporangia， sporangia pedicels and other morphological characters（Gallegly and Hong， 2008）. It makes classification and identification of Phytophthora species become difficult， given that the morphological characteristics of Phytophthora will be varied with different culture conditions， and Phytophthora species have a great variability themselves. With the application of molecular biology in classification and identification of Phytophthora species， many reports have confirmed that 5.8S rDNA ITS region of Phytophthora is a stable conservative region and has specificity of interspecies as well. So that primers can be designed in the specific region of ITS for molecular identification of Phytophthora（Cooke and Duncan， 1997； Wang et al.， 2000； Li et al.， 2007b； Cheng et al.， 2014）. P. colocasiae was identified as pathogen of leaf blight of Lipu Taro using methods of morphology and molecular biology in this study. The ITS sequence obtained in our study showed 99% of homology to that of P. colocasiae isolates of taro leaf blight in NBCI. All ITS sequences of P. colocasiae isolated from Guangdong， Guangxi， Hunan and Hubei in China showed 97%-100% identical to isolates of P. colocasiae in GenBank database（Zhou et al.， 2012）， which were similar with the results reported by Lu et al.（2013）， who also observed that all of isolates collected from south-eastern Guangxi were A2 mating type. It remains unclear as which mating type of P. colocasiae that the present isolate belongs to. Wang et al.（2001） reported that rich genetic diversity of P. colocasiae strains collected from Hainan province， based on the significant differences in morphological characteristics， biology and virulence of 19 P. colocasiae isolates. The comparative results of P. colocasiae strains between Guangxi and Hainan revealed that evident differences were found in their biological characteristic and pathogenicity（Ye et al.， 2016）. According to the above-mentioned analysis， we speculated that high genetic variation present within population of P. colocasiae in China.

An efficient， stable genetic transformation system needs to be developed before genetic manipulation and function analysis of plant pathogen becomes possible. But the available methods for genetic transformation of fungi are not suitable for transforming oomycetes species， partially because that oomycetes evolved independently from fungi. A transformation protocol mediated by the polyethylene glycol（PEG） has been developed to introduce foreign DNA into Phytophthora （Judelson et al.， 1991）. This transformation system has been successfully used for the expression of exotic genes in P. megasperma f. sp. glycinea（Judelson et al.， 1993）， P. nicotianae（Bottin et al.， 1999）， P. palmivora（van West et al.， 1999） and P. brassicae（Si-Ammour et al.， 2003）. In addition， green fluorescent protein （gfp） has been expressed in P. palmivora（van West et al.， 1999）. Preparation of protoplasts is the first and a necessary step in PEG-mediated transformation system. In this study， protoplasts of P. colocasiae were generated following protocol as described（McLeod et al.， 2008）. However， genetic transformation system for P. colocasiae hasnt been established or tested， and would be our attempt in the further study.

4 Conclusion

It concluded that the causal pathogen of taro leaf blight in Lipu of Guangxi is identified as P. colocasiae and its prepared protoplasts could be used for further study of taro leaf blight.

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（責(zé)任編輯 韋莉萍）