lsolation and ldentification of Fungus Causing Collar Rot Disease of Rubber Tree

Guizhi JlANG，Yongfang WANG，Ming ZHOU，Guohua LlYunnan Institute of Tropical Crops/Xishuangbanna Comprehensive Experimental Station of National Natural Rubber Industry Technology System，Jinghong 666100，China

lsolation and ldentification of Fungus Causing Collar Rot Disease of Rubber Tree

Guizhi JlANG，Yongfang WANG，Ming ZHOU*，Guohua Ll
Yunnan Institute of Tropical Crops/Xishuangbanna Comprehensive Experimental Station of National Natural Rubber Industry Technology System，Jinghong 666100，China

Collar rot caused the death of Hevea rubber in a village located in Jino County of Xishuangbanna in September，2012.In order to elucidate the causal agent of collar rot and its taxonomic status，the causal fungus was isolated and identified through a series of tests about its pathogenicity，morphology，culture feature and biological characteristics.The results indicated that the fungus was Fusarium venfricosum.It could grow at temperatures ranging from 10 to 40℃，and the most suitable temperature was 30℃.Its mycelium growth was inhibited above 40℃ or below 10℃.The accumulated water in wet season was the main reason for this disease occurrence.

Rubber；Disease；Collar rot；Fusarium

O n September 25th，2012，an investigation of collar rot which caused the death of 7-year-old Hevea rubber was carried out in a village located in Jino County of Xishuangbanna.The symptoms showing up were completely different with previously reported collar rot caused by Phytophthora and Fusarium oxysporum. There appeared powdery mildew on the trunk adjacent to earth in affected trees.The bark covered by mildew was brown necrosis，and the necrotic spot was quite clear.After removal of this bark，the affected xylem part was found with white gel and no extraneous odor.During the late period，the bark got sink and resulted in death of the whole tree（Fig.1）.It was in site diagnosed that this should be a new rubber collar rot，sample was then collected to lab for pathological observation and pathogen identification，the results are reported as follows.

Materials and Methods

Pathogen isolation

Samples collected from field were washed and dried，and the part in the junction of disease and health was cut off by a size of（2-3）mm×（2-3）mm with a knife，sterilized in disinfectant of 0.1%arsenic and mercury for 1 min，and washed three times with sterile water.After that，the infected tissue was inoculated into a PDA plate，cultured at 25 to 30℃for 5 d.The representative colonies were future isolated and picked up for pathogenicity identification［1］.

Pathogenicity identification

Two-year-old rubber seedlings were selected as explants and surface sterilized with 75%ethanol before inoculation.The stem base was inoculated with the purified strain using a needle.The inoculated part was wrapped by plastic film，and cultured at temperature of 25 to 30℃naturally for 7 d.15 d later，the affected material was collected to isolate the pathogens which were then cultured at room temperature of 25 to 30℃for 4 to 5 d before the morphological characters of the colonies were observed and compared with those isolated from necrotic tissue.

Morphological characters and identification

The isolated spore was inoculated into PDA plates and culture for 5 to 7 d，before the morphological characters were observed through a microscope and photographs were taken when the colony was formed.Species identification was conducted based on its colony and conidium morphology.

Determination of suitable growth temperature

The pathogen isolated in above steps was inoculated into a PDA plate and cultured for 5 d，before round colonies in diameter of 5 mm were punched by a sterilized puncher.The inoculated masses in PDA plates were cultured at temperatures of 10，15，20，25，30，35 and 40℃respectively for 5 to 7 d，with relative humidity of 80%.

Screening of fungicides for pathogen control in laboratory

The cultured strain was inoculated and cultured in media supplemented with thiram， chlorothalonil， thiophanate-methyl，mancozeb and carbendazol at room temperature of 25 to 30℃for 5 d，before the observation of colony growth in each toxic medium.

Results and Analysis

Pathogen isolation and pathogenicity identification

After cultured for 5 d，four types of colonies formed，and the preponderant one was selected and transferred to a PDA plate，cultured for another 5 d. The isolated strains were inoculated to the stem base of two-year-old rubber seedlings.There appeared symptoms similar with that of trees in field 15 d later.The pathogen was isolated from affected tissue and cultured at room temperature of 25 to 30℃for 4 to 5 d. The morphological characters in colony were in coincidence with that of isolated from necrotic tissue，confirming this should be the same fungi causing the rubber collar rot in field.

Morphological characters in colony and species identification

Colony morphological characteristics The fungus isolated from the single spores of the confirmed pathogen was cultured in PDA plate for 5 d for observation of morphological characters.Its aerial mycelium was flocculent，grey white，yellow brown in base and turned to brown after 10 d of culture on PDA plate（Fig.2）.

Morphology and growth mode of conidia The strain was grey white at 25 to 30℃in natural light，sprouting，with septates in width of 1.84 to 4.10滋m.Aggregated spores grew on mycelium （Fig.3A）.The first conidiophore was upright，with no sprouting，bottle-shaped sterigma on the top.After the first conidium appeared on the sterigma （Fig.3B），more conidia keep growing in the inner wall and stayed on the outer wall of the top sterigma（Fig.3C）.The conidia finally broke，some of then adhered to the gum drops（Fig.3E）.The large conidia was straight，or irregularly bended，long oval-shaped，blunt on the top，with obvious sertoli cell，with 3 to 6 septates in size of 5.30 to 6.8滋m ×11.51 to 18.10滋m.The small conidia shaped in oval or obovate，some bended irregularly，with 0 to 1 septate in size of 2.12 to 3.68滋m×4.27 to 11.16滋m（Fig.3E）. In aged medium，paralleled mycelia showed block-like， chlamydospore was single or aggregated in 2 or 3，round or nearly round，with thick and smooth wall，6.6 to 8.70滋m in diameter，formed in the end of short side sprouting（Fig.3F）.

This strain was identified as F.venfricosum based on the morphological characters and sproulating ways of conidia［3-5］.

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腹?fàn)铉牭毒鸬南鹉z樹基腐病的分離鑒定

蔣桂芝，王勇芳，周 明*，李國華 （云南省熱帶作物科學(xué)研究所/國家天然橡膠產(chǎn)業(yè)技術(shù)體系西雙版納綜合試驗(yàn)站，云南景洪 666100）

橡膠樹因基部腐爛死亡，經(jīng)對(duì)其病原菌的分離、致病性測定，根據(jù)病原菌菌落形態(tài)特征，鑒定病原菌為鐮刀菌屬腹?fàn)铉牭毒‵usarium venfricosum）。該病菌的適宜生長溫度為10～40℃，最適生長溫度為30℃，在40℃以上或10℃ 以下，菌絲的生長受到抑制。導(dǎo)致這次病害發(fā)生的主要原因是雨季積水。

橡膠樹；病害；基腐；鐮刀菌

云南省科技創(chuàng)新強(qiáng)省計(jì)劃（2009AB001）； 國 家 科 技 支 撐 計(jì) 劃（2011BAD30B01）；國家天然橡膠產(chǎn)業(yè)技術(shù)體系西雙版納綜合試驗(yàn)站項(xiàng)目（nynytx-34-zd5）。作者簡介蔣桂芝（1967-），女，貴州天柱人，從事熱帶植物保護(hù)研究，高級(jí)農(nóng)藝師，E-mail：316606049@qq.com。*通訊作者，副研究員，從事熱帶植物保護(hù)，E-mail：xsbnzhouming@126. com。

2014-11-25

2015-01-20

Supported by Planned Project for Strengthening Scientific and Technology Innovation of Yunnan Province （2009AB001），National Key Technology Research and Development Program （2011BAD30B01）；Fund from Xishuangbanna Comprehensive Experimental Station of National Natural Rubber Industry Technology System（nynytx-34-zd5）

.E-mail:xsbnzhouming@126.com

November 25，2014Accepted:January 20，2015