Title：
First Report of Diaporthe lithocarpus Causing Postharvest Rot of Kiwifruit in Sichuan Province, China
Authors：
Corresponding
Author：
L. Li, H. Pan, M. Y. Chen, and C. H. Zhong
Pubyear：
2016
Title of
Journal：
Plant Disease
Paper
Code：
Volume：
100
Number：
11
Page：
2327
Others：
Classification：
Source：
Abstract：
The prevalence of kiwifruit (Actinidia sp.) postharvest rot disease in China was studied from October to December 2014. Approximately 30% of fruits were observed with milky-white, sour-smelling, watery internal tissue. To identify the causal agent of the disease, 100 fruits (cvs. Jinyan, Hongyang, Jinkui, Guichang, and Qinmei) were arbitrarily collected from Hubei, Sichuan, Henan, Chongqing, Jiangxi, Guizhou, and Shaanxi provinces and stored in controlled-atmosphere rooms (4°C, 2% O2, 5% CO2). After 60 days, 28 fruit were observed to have postharvest rot. Botryosphaeria dothidea and Diaporthe nitschke (anamorph: genus Phomopsis), the two most important postharvest fungal pathogens of kiwifruit in China (Li et al. 2015; Zhou et al. 2015), were isolated from most fruits. Most Diaporthe sp. strains were identified morphologically and molecularly as D. ambigua, previously reported to cause rot in stored kiwifruits in Chile (Auger et al. 2013). Five previously unidentified Issue Date: 13 Oct 2016 Published: 17 Aug 2016 First Look: 21 Jun 2016 Accepted: 16 Jun 2016 1/2 morphologically identical strains were solated from cv. Jinyan, from Pujiang County, Chengdu, Sichuan Province, and characterized by confocal optical microscopy at 400× magnification. Two types of conidia were observed: α-conidia 6.2 to 7.8 × 2.3 to 2.9 μm, unicellular, aseptate, fusiform, hyaline, biguttulate, tapering toward both ends; β-conidia 18.3 to 26.5 × 1.03 to 1.72 μm, aseptate, eguttulate, filiform, curved or hamate, with obtuse ends. Morphologically, these matched the holotype strains CGMCC 3.15175 of D. lithocarpus (Speg.) (Gao et al. 2014). To confirm identification, three isolates (KFRD-3, KFRD-5, KFRD-6) were sequenced. The rDNA internal transcribed spacer (ITS) and beta-tubulin (BT) regions were amplified using ITS4/ITS5 and Bt2A/Bt2B primers, respectively (Hu et al. 2007). BLASTn was performed, and maximum likelihood trees based on the concatenated nucleotide sequences of the two genes were constructed using MEGA7. The ITS sequence amplified (GenBank KR703276, KX249709, and KX249710) shared 99% identity with the paratype of D. lithocarpus CGMCC 3.15177, 3.15179 (GenBank KC153100, KC153101). The BT sequence amplified (GenBank KU377339, KX249711, and KX249712) shared 99% identity with the paratype of D. lithocarpus CGMCC 3.17100, 3.17101, 3.17095 (GenBank KF576303, KF576304, and KF576309). Molecular phylogenetic analysis confirmed the three strains were D. lithocarpus. Pathogenicity tests on 20 mature surface-disinfested, A. chinensis (cv. XinGuan No 2) fruits involved creating small wounds in the peel in both the upper and lower areas of 10 fruit using a sterile scalpel. The upper area of five fruits was inoculated by filling the wound with a solid agar plug (5 mm) infested with actively growing D. lithocarpus, while the other five fruits received 10 μl injections of a spore suspension (1 × 106 conidia/ml) of D. lithocarpus. The bottom wound area (negative control) on each fruit received either 10 μl sterile agar or sterile water. Ten unperforated fruits were inoculated by the same method. Fruit were incubated at 22/18°C (day/night), 80% humidity. After 6 days, typical symptoms of postharvest rot, with softened areas 6.5 to 7.2 mm diameter were observed at wound sites of both inoculation methods (plug and solution). Experiments were repeated three times and the fungus was reisolated each time and identified as D. lithocarpus by morphological and molecular analysis. Noninoculated and nonwounded fruits showed no symptoms. This is the first report of kiwifruit postharvest rot caused by D. lithocarpus in China. To prevent the spread of kiwifruit rot diseases, more care should be taken to avoid wounding during or after harvest.
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