Identification of Pathogens on Dry Blight Disease in Leaf Tip of Red-fleshed Kiwifruit in Qiandongnan Prefecture

Guohui ZHANG Xiangyang LI Huanxian GU Wenhua ZHANG Shengli SI

Abstract In order to reduce the harm of diseases in red-fleshed kiwifruit in Qiandongnan Prefecture and ensure product quality, a new disease in the producing areas were investigated, and pathogen identification were carried out. The pathogen was isolated from diseased leaves by a tissue separation method. DNA sequencing was performed by the sequence analysis of ribosomal rDNA-ITS (Internal transcribed spacer) region, and a molecular evolutionary tree was constructed by the MEGA 4.0 software. The pathogenic fungus was classified and identified by combining morphological observation finally. The results showed that the dry blight disease in leaf tip was a new disease, which was caused by Epicoccum sorghinum, and it had a serious damage to red-fleshed kiwifruit.

Key words Red-fleshed kiwifruit; Dry blight disease in leaf tip; Epicoccum sorghinum; Disease symptom; Pathogen identification

Received: July 8, 2022  Accepted: September 9, 2022

Supported by Province, Prefecture and School Science and Technology Cooperation Agreement Project of Science and Technology Department of Guizhou Province (QKH H Z［2017］7178); The Science and Technology Foundation Project of Guizhou Province ( 2017-1166).

Guohui ZHANG (1978-), female, P. R. China, professor, PhD, devoted to research about phytopathology and agricultural product quality and safety.

*Corresponding author. E-mail: 59833259@qq.com.

Red-fleshed kiwifruit is a kiwifruit variety in Actinidia chinensis Planch. It is an early-ripening red-fleshed variety and a rare variety for both medicine and food. It has health care effects including nourishing blood, beautifying skin, expelling toxins and preventing cancer, and it has adjuvant effects on Vc deficiency, hypertension, cardiovascular and cerebrovascular diseases and cancer［1-5］. Red-fleshed kiwifruit has become a pillar industry for farmers in Qiandongnan Prefecture, Guizhou Province to increase their income. However, farmers lack knowledge of kiwifruit diseases and scientific prevention and control techniques, resulting in increasingly serious orchard diseases in some areas, affecting the healthy development of the local kiwifruit industry. In 1998, Huang and Qi first reported on the root rot of kiwifruit in Guangdong Province by means of routine separation of diseased tissues［6］. Since then, kiwifruit brown spot［7-11］, anthracnose［12-15］, black spot［16］, yellowing disease［17-20］, bacterial leaf blight［21-24］, fungal diseases such as soft rot［25-26］ and root-knot nematode［27-30］, among which brown spot, anthracnose, bacterial leaf blight, black spot and yellowing are all leaf diseases, accounting for the largest proportion of reported diseases. At present, there has been no research report on dry blight disease in leaf tip of red-fleshed kiwifruit at home and abroad. The damage of this disease to red-fleshed kiwifruit is far greater than that of other leaf diseases, and severe cases can lead to the death of the whole plant. In this study, the symptoms of dry blight disease in leaf tip of red-fleshed kiwifruit were investigated, and the pathogen was identified by collecting specimens and conducting indoor pathogen analysis, aiming to supplement diseases of red-fleshed kiwifruit and provide support for the later disease control.

Materials and Methods

Experimental materials

Disease specimens

From October 2019 to May 2020, diseased plants were collected from the red-fleshed kiwifruit planting base in Banpo Group, Cenhua Village, Huangping County, Qiandongnan Miao and Dong Autonomous Prefecture, Guizhou Province, and the symptoms of the disease were recorded. Fresh specimens were collected at the Inspection and Quarantine Station of Kaili Forestry Bureau for the isolation and identification of pathogens.

PSA medium

Potatoes were peeled and cut into pieces, and 200 g was weighed, and boiled in 1 000 ml of boiling water for 20 min. After filtration, the filtrate was added with 15 g of sucrose, and 17 g of agar powder, and water was finally added to 1 000 ml, which was subpackaged into conical flasks, which were sterilized in an autoclave for 20-30 min.

Instruments and reagents

The instruments used in the experiment included an autoclave, constant temperature incubators, microscopes, an ultra-clean workbench, transplant needles, scissors, tweezers, induction cookers, microwave ovens, etc. The reagents used included 75% ethanol, 0.1% mercuric chloride solution, sterile water and industrial ethanol.

Experimental method

Symptom observation and description

The lesion shape, color, disease site and damage degree of red-fleshed kiwifruit were observed and described, and the disease sites were photographed at the same time.

Pathogen isolation, purification and identification

The diseased leaves and stems of red-fleshed kiwifruit were separated by a tissue separation method, that is, three pieces of diseased tissue were cut with scissors, and soaked in 0.1% mercury chloride solution for 2-3 min and then in 75% ethanol for 2-3 min, and then, the soaked tissue was rinsed with sterile water for 3 times, and finally picked up with tweezers and placed in the PSA solid medium in the shape of a "pin". The plates were placed upside down in a constant temperature incubator at 25 ℃ for 5-7 d. The grown colonies were purified twice by transplant needles until a pure culture was obtained. When purified colonies covered all over the medium, a spot of the hyphae was picked from the central part of a colony and placed on a glass slide, and pictures were taken under a microscope for the typical morphological characteristics of the pathogenic fungus to observe, measure and record the spore size and hyphae at the same time.

The sequence analysis method of ribosomal rDNA-ITS (Internal transcribed spacer) region was adopted for DNA sequencing. The sequencing results of the ITS region sequence were compared by BLAST in the GenBank database, and the MEGA 4.0 software was used to construct a molecular evolutionary tree. Finally, the pathogen was classified and identified based on the morphological observation results.

Pathogen inoculation

The purified pathogen was first cultured on PSA medium for 5 to 7 d, and the spores were flushed off and prepared to a spore suspension with a concentration of 106 spores/ml. Fresh leaves of red-fleshed kiwifruit were inoculated with the spore suspension by a puncturing method and a spray method. After typical lesions had appeared on leaves, according to Koch’s rule, the lesions were separated and purified by the tissue separation method, and finally, the isolated pathogens were identified.

Results and Analysis

Observation and description of symptoms

In the early stage of diseased leaves of red-fleshed kiwifruit, dark brown discoloration and withering appeared on the front of the leaf tip. Later, with the aggravation and spread of the disease, the leaf tip lesions spread to the petiole in a large area, and the diseased part showed a large area of dark brown withered necrosis. The junction of diseased and healthy tissues at the diseased site was obvious, and the edges of the lesions were mostly irregular. When the disease of kiwifruit leaves was severe in the later stage, the lesions could span the veins, and 2/3 of the leaves showed large brown necrotic spots, and the front of the leaves was withered and curled (Fig. 1a). Diseased leaves of red-fleshed kiwifruit were dark gray-brown at the back, and showed dark brown veins. The junction between the diseased and healthy tissues was obvious, and the leaves were curled and shriveled (Fig. 1b). In addition, the appearance of branches where the diseased leaves were located was normal, but it could be easily broken by lightly folding, and the broken stem showed clear annular browning, that is, browning of vascular bundles (Fig. 1c).

Pathogen isolation, purification and identification

After culturing on PSA medium at 25 ℃ for 7 d, the edges were regular and round, and the hyphae were gray and felt-like, and the center of each colony had small white and orange-red circular protrusions (Fig. 1d). The pathogen could produce two kinds of spores, chlamydospores and conidia. The chlamydospores were clustered or solitary, colorless, and the size was (6.98-20.45) μm×(5.38-18.26) μm (Fig. 1e). The conidia were oblong or long oval, single-celled, colorless, and the size was (0.9-2.9) μm×(0.9-1.2) μm (Fig. 1f). Morphological observation and ribosomal rDNA-ITS (internal transcribed spacer) region sequence analysis were used for identification. The sequencing result of the ITS region were compared by BLAST in the GenBank database, and according to the construction results of the molecular evolutionary tree (Fig. 2), the strain was clustered with Epicoccum sorghinum into a group (Fig. 2), so the pathogen was determined to be E. sorghinum. In view of the fact that E. sorghinum could cause symptoms such as branch blight and leaf spot, and combined with the disease characteristics of red-fleshed kiwifruit, it was tentatively named dry blight disease in leaf tip of red-fleshed kiwifruit.

Pathogen inoculation

According to Koch’s rule, the prepared spore suspension was inoculated on the leaves of red-fleshed kiwifruit by a puncturing method. After 3 d, the leaves of red-fleshed kiwifruit began to develop diseased spots, and after 4 d, the diseased spots covered the whole leaves, and the diseased leaves were dark brown, withered and shrunken (Fig. 3). The pathogen on the lesions was isolated by the tissue separation method, and microscopic identification was carried out. The results showed that the strain isolated again was the same as the inoculated pathogenic strain, which proved that the inoculated strain was the pathogen of dry blight disease in leaf tip of red-fleshed kiwifruit.

Conclusions and Discussion

The red-fleshed kiwifruit industry in Qiandongnan Miao and Dong Autonomous Prefecture of Guizhou Province plays an important role in local economic development and farmers’ income generation. In order to reduce economic losses, a disease investigation was carried out on the current important disease in the production area. The results showed that the symptoms of dry blight disease in leaf tip of red-fleshed kiwifruit caused by E. sorghinum were serious, which was the first report in China.

The results of this study showed that the symptoms of dry blight disease in leaf tip of red-fleshed kiwifruit in the red-fleshed kiwifruit planting base in Banpo Group, Cenhua Village, Huangping County, Qiandongnan Prefecture, Guizhou Province were serious, which led to a large area of withered leaves and produced browning of vascular bundles at the stem. The pathogen was E. sorghinum, a fungus belonging to the genus Epicoccum, which can cause stem blight, branch blight and fruit rot in various crops, thus seriously affecting the growth and development of the affected plants［31］. As for the pathogen reports of dry blight disease in leaf tip, past research reports are mostly about Phoma lilii［32］, Botryosphaeria dothidea［33］ and Phyllosticta oryzicola［34］. In this study, the above three types of pathogens were not isolated, only

E. sorghinum was isolated, and the identification method of morphological microscopy combined with molecular biology was used. At present, there are few reports on E. sorghinum at home and abroad. Existing data showed that E. sorghinum can not only damage crops as a pathogen, but also has antibacterial activity as an endophyte［35］. Epicoccum is an important class of phytopathogenic fungi belonging to the family Didymellaceae［36-37］, which can infect commercial crops, agricultural crops, forest trees, weeds, etc.［38-39］, causing stem wither, base rot, leaf spot, branch withering, fruit rot and other symptoms of various crops such as Pennisetum hydridum［40］, dragon fruit［41］, apple［42］, and bilberry［43］, severely affecting growth and development of infested plants. No control test was conducted in this study, but Zeng et al.［31］ reported in detail the dry blight disease in leaf tip of lily caused by E. sorghinum, and suggested that boscalid had the strongest inhibitory effect on E. sorghinum. Considering that E. sorghinum can cause fatal damage to kiwifruit plants, boscalid can be used as an alternative for the control of dry blight disease in leaf tip of red-fleshed kiwifruit.

References

［1］ HUANG DR. Brief introduction of the cultivation techniques of kiwifruit［J］. Shanxi Agricultural Economy, 2018(14): 80-81. (in Chinese).

［2］ LUO GF. Analysis of the diseases and pests of kiwifruit and their control［J］. Agriculture of Jilin, 2012(1): 57-58. (in Chinese).

［3］ MA R, LIU BH, LI J, et al. Main types of diseases and pests in red-fleshed kiwifruit in different growth stages and their control techniques［C］//Proceedings of 2018 Annual Academic Meeting of Yunnan Plant Protection Society-Green Plant Protection Supporting Plateau Characteristic Agriculture. Kunming: Yunnan Agricultural Science and Technology Editorial Department, 2018: 105-106. (in Chinese).

［4］ ZHANG ZH, LIU L. Analysis of red-fleshed kiwifruit and its cultivation techniques［J］. Agricultural Technical Services, 2017, 34(16): 85, 89. (in Chinese).

［5］ ZHU XL, ZHU ZL. Integrated control technology of main diseases and pests of kiwifruit［J］. Agriculture and Technology, 2019, 39(16): 133-134. (in Chinese).

［6］ HUANG YJ, QI PK. Study on the etiology of kiwifruit root rot in Guangdong Province［J］. Journal of South China Agricultural University, 1998(4): 22-25, 38. (in Chinese).

［7］ ZHAO JM. Identification and research on the chlorine dioxide efficacy of pathogens causing brown spot disease on Actinidia chinensis［D］. Xi’an: Shaanxi Normal University, 2014. (in Chinese).

［8］ ZHAO JM, GAO GT, GU LJ, et al. Identification and pharmaceutical screening of brown spot disease on Actinidia chinensis［J］. Scientia Agricultura Sinica, 2013, 46(23): 4916-4925. (in Chinese).

［9］ LYU PK, SU HL, GAO ZJ. Primary color illustration of control of diseases and pests in kiwifruit, wolfberry and fig［M］. Beijing: Chemical Industry Press, 2014: 5-7. (in Chinese).

［10］ WU XZ. Experiment on chemical control of kiwifruit brown spot［J］. Modern Horticulture, 2018(15): 45-46. (in Chinese).

［11］ PAN H, HU QL, ZHANG SJ, et al. Kiwifruit disease investigation and pathogen identification in Liupanshui City, Guizhou Province［J］. Plant Protection, 2018, 44(4): 125-131,137. (in Chinese).

［12］ YAN PS, YAN YZ. Occurrence regularity and comprehensive control of kiwifruit anthracnose［J］. Xibei Yuanyi, 2014(1): 27-28. (in Chinese).

［13］ PAN H, DENG L, CHEN MY, et al. Annual investigation and pathogen identification of kiwifruit disease in Taishun County, Zhejiang Province［J］. Jiangsu Agricultural Sciences, 2019, 47(14): 104-111. (in Chinese).

［14］ ZHAO F. Identification and control of pathogens of Collectotrichum sp. on kiwifruit stems in Fengxin County［D］. Nanchang: Jiangxi Agricultural University, 2013. (in Chinese).

［15］ ZHANG JP. Kiwifruit leaf diseases becoming increasingly serious in recent years［J］. Pesticide Market News, 2017(27): 67. (in Chinese).

［16］ LIU G. Wuhan Botanical Garden of Chinese Academy of Sciences identifies and publishes three kiwifruit disease pathogens［J］. Pesticide Market News, 2018(30): 54. (in Chinese).

［17］ ZHANG GY, SONG Y. Analysis and solution of kiwifruit yellowing problem［J］. Xibei Yuanyi, 2020(1): 38-40. (in Chinese).

［18］ LIU WG, WANG F, ZHAO Q, et al. Effects of different fertilizers on control of kiwifruit etiolated disease and yield［J］. Journal of Anhui Agricultural Sciences, 2018, 46(27): 154-156,165. (in Chinese).

［19］ ZHANG JJ, WANG XQ. Preliminary report on the trial of "Tianbang Shikelu" for controlling kiwifruit yellowing disease［J］. Shaanxi Journal of Agricultural Sciences, 2002(5): 29-30. (in Chinese).

［20］ LIU XF, FAN XF, ZHANG LS, et al. Inducing factors of iron deficiency chlorosis of kiwis in Guanzhong area of Shaanxi［J］. Acta Agriculturae Boreali-occidentalis Sinica, 2002(2): 57-59. (in Chinese).

［21］ FU B, WANG JZ, ZHANG F, et al. Screening and identification of broad-spectrum antagonistic strains of kiwifruit leaf blight pathogens［J］. Shaanxi Journal of Agricultural Sciences, 2019, 65(10): 22-26. (in Chinese).

［22］ SONG XB, ZHANG XW, HUANG J. Studies on pathogen and occurrence factors of the bacterial withered leaf on kiwifruit［J］. Journal of Northwest A & F University: Natural Science Edition, 2003(5): 73-76. (in Chinese).

［23］ LI J, CHEN L, CHEN L, et al. Selection of bacillus strains antagonistic to kiwifruit bacterial leaf blight disease and their biocontrol effect in fields［J］. Chinese Journal of Biological Control, 2010, 26(2): 235-239. (in Chinese).

［24］ LI J. Biological control technology and field application of common diseases of kiwifruit in Shaanxi［D］. Xi’an: Northwestern University, 2010. (in Chinese).

［25］ VIDHYA PALLAVI R, NEPOLEAN P, BALAMURUGAN A, et al. In vitro studies of biocontrol agents and fungicides tolerance against grey blight disease in tea［J］. Asian Pacific Journal of Tropical Medicine, 2012, 2(1): S435-S438.

［26］ LIU N, XIE GF. Analysis of fungal diversity during the pathogenesis of ‘Guichang’ kiwifruit soft rot［C］//Abstract Collection of Papers of the 16th Annual Meeting of Chinese Institute of Food Science and Technology and the 10th China-US Food Industry Forum. Beijing: Chinese Institute of Food Science and Technology, 2019: 427-428. (in Chinese).

［27］ CHANG Q, LI YM, YANG YW, et al. Occurrence status of kiwifruit root-knot nematode disease in Shaanxi Province［C］//Proceedings of the 2019 Annual Academic Conference of China Society of Plant Protection. Beijing: China Society of Plant Protection, 2019: 98-103. (in Chinese).

［28］ CHEN W, SUN YF, WU SP, et al. Occurrence status and species identification of root knot nematode endangering kiwifruit in Xiuwen County, Guizhou［J］. Southwest China Journal of Agricultural Sciences, 2018, 31(1): 84-88. (in Chinese).

［29］ LI SJ, YU Z. A new species of kiwifruit root-knot nematode from Henan (Meloidogyne actinidiae)［J］. Journal of Henan Agricultural University, 1991(3): 251-254. (in Chinese).

［30］ LIU C, WANG CG, ZHANG F, et al. Identification of root-knot nematode on Actinidia chinensis in Zhouzhi County［J］. Modern Agricultural Science and Technology, 2018(2): 117-118. (in Chinese).

［31］ ZENG HL, LU QN, ZENG PY, et al. Identification, biological characteristics and sensitivity of the causal pathogen inducing leaf dieback on lily［J］. Acta Horticulturae Sinica, 2018, 45(12): 2407-2416. (in Chinese).

［32］ TANG XN, LIU FX, XIAO AP, et al. Investigation and identification of lily diseases in Jiangxi［J］. Acta Agriculturae Jiangxi, 1997, 9(4): 1-8. (in Chinese).

［33］ BEN HY. Identification of major ornamental plant diseases in North China and the Discovery of new diseases［D］. Harbin: Northeast Agricultural University, 2008. (in Chinese).

［34］ XU JY, WANG ZM, TONG YH. Rice phyllosticta leaf blight phylloslicta oryzicola rice［J］. Acta Phytopathologica Sinica, 1995(1): 9-11. (in Chinese).

［35］ ZHU X. Isolation of Epicoccum sorghinum and identification of resistance in multigene transgenic maize lines［D］. Wuhan: Huazhong Agricultural University, 2018. (in Chinese).

［36］ TAI LM, ZHENG W, YAN FY, et al. Studies on fungi of rice seeds［J］. Journal of Heilongjiang Bayi Agricultural University, 2003, 15(1): 31-33. (in Chinese).

［37］ LI W, CHEN Y, ZHANG XX, et al. Pathogenic fungi of wheat brown foot rot and their pathogenicity in China［J］. Journal of Triticeae Crops, 2011, 31(1): 170-175. (in Chinese).

［38］ STOKHOLM MS, WULFF EG, ZIDA EP, et al. DNA barcoding and isolation of vertically transmitted ascomycetes in sorghum from Burkina Faso: Epicoccum sorghinum is dominant in seedlings and appears as a common root pathogen［J］. Microbiological Research, 2016(191): 38-50.

［39］ LIU P, WEI M, ZHU L, et al. First report of leaf spot on taro caused by Epicoccum sorghinum in China［J］. Plant Disease, 2018, 102(3): 682.

［40］ LIAN QX, SANG WJ, LI XX, et al. Investigation and control of fungal diseases of Pennisetum sinese Roxb in Huajiang area of Guizhou［C］//Plant Protection and Modern Agriculture: Proceedings of the 2007 Annual Academic Conference of China Society of Plant Protection. Beijing: China Society of Plant Protection, 2007: 225-228. (in Chinese).

［41］ WANG JL, REN JG, LIU HM. Identification of the pathogen causing pitaya stem disease［J］. Chinese Agricultural Science Bulletin, 2014, 30(10): 307-311. (in Chinese).

［42］ HOU YM. Etiological study of black spot disease on bagged fruit of Fuji apples［D］. Yangling: Northwest A&F University, 2016. (in Chinese).

［43］ WEI YY, LIANG C, ZHAO HH, et al. Pathogen identification and their diversity of blueberry postharvest diseases in east China［J］. Shandong Agricultural Sciences, 2017, 49(7): 112-115. (in Chinese).

［44］ BRUTON BD, REDLIN SC, COLLINS JK, et al. Postharvest decay of cantaloupe caused by Epicoccum nigrum［J］. Plant Disease, 1993, 77(10): 1060-1062.