Cui Lina, Zou Chengjia, Zhang Zhenyu, Duan Luyao, Huang Jimei, Wang Liming, Xiao Weihua, Yang Xiaorong, Xiang Yunjia, Li Wenyi, Li Xiao, Zhang Haiyan
Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Chengdu, Sichuan, China;
Institute of Plant Protection, Sichuan Academy of Agricultural Sciences, Chengdu, China;
Plant Dis. 2022 Jun 9. doi: 10.1094/PDIS-01-22-0152-PDN.
Maize (Zea mays L.) is one of the most important crops in China. Since 2020, a new leaf spot disease has occurred in southwest China in areas such as Yunnan, Sichuan and Hubei provinces. Typical symptoms appeared after tasseling. The spots are scattered on the leaf surface, round to oval in shape with diameter range 3-20 mm. Spots are initially water-soaked, gradually turning yellow or white. In 2021, diseased leaf samples with typical white spot were collected for pathogen isolation and identification in Qujing, Yunnan province. Four small pieces of leaf tissue (about 0.25 cm2 in area) were excised from the edge of the necrotic lesion of each plant, surface sterilized with 75% ethanol for 1 min, rinsed three times with sterile distilled water, and soaked in sterile distilled water for 5 min. The solution was plated on Luria Broth medium (LB) plate (Shin et al. 2022) . After incubation at 28°C for 24 h, round, smooth-edged yellow colonies appeared in the LB plate. The bacterium isolated was gram-negative, negative for oxidase, positive for peroxidase, indole, citrate (Wells et al. 1987). Three strains (PA21QJ01, PA21QJ02 and PA21QJ03) showed identical colony morphology. PA21QJ01 was used for further molecular analyses. DNA was extracted from fresh colonies cultured in LB(Shin et al. 2022), and the fragments at the 16S rDNA, gyrB and rpoB loci were amplified using primers 27F/1492R (Galkiewicz and Kellogg 2008), UP-1/UP-2r (Yamamoto and Harayama 1995) and rpoBCM81-F/rpoBCM32b-R (Brady et al. 2008), respectively. The sequences of fragments of 16S rDNA, gyrB and rpoB from isolate PA21QJ01 were was deposited in GenBank (accession number: OM184301.1, OM302500, OM302499). A search for homologous sequences using BLAST resulted in 99.9, 99.6 and 99.8% identity of 16S rDNA, gyrB and rpoB, respectively, with sequences from the NN08200 of Pantoea ananatis (GenBank accession numbers: MK415050.1 for 16S rDNA, CP035034.1 for gyrB and CP035034.1.1 for rpoB). Above molecular results indicated that PA21QJ01 isolated from maize white spot is P. ananatis. Pathogenicity tests were performed on tasseled plants of the suscptible maize variety Wugu1790. After culture in LB medium plate at 30°C for 12 h, the bacterial solution was used for inoculation at a concentration of 1 × 108 CFU ml-1. After 7 days of inoculation, the leaves of the plants appeared water-soaked. After 10 days, white spot developed with brown margin. In contrast, the control plants remained healthy and symptomless. The same P. ananatis was reisolated in the inoculated maize plants, fulfilled Koch's law. In the last decade, P. ananatis has been reported to cause maize white spot in South Africa, Mexico, Poland, Argentina, Brazil (Sauer et al. 2015), and Ecuador (Toaza et al.2021). It has caused crop diseases with other crops, such as onion, rice, pineapple, melon, and sorghum, and others (Sauer et al. 2015). It caused leaf blight and leaf steak in rice in China (Yu et al. 2021). This is the first report of maize white spot caused by P. ananatis in China. However, to our knowledge, this is the first report of maize white spot disease in China. Attentions should be paid to screening for disease-resistant resources and breeding disease-resistant hybrids. Reference: Wells, J. M. et al. 1987. Int. J. Syst. Bacteriol. 37:136-143. Shin, G. Y. et al. 2022. Plant Dis. Doi: 10.1094/PDIS-08-21-1810-SC. Brady, C., et al. 2008. Syst. Appl. Microbiol. 31:447. Galkiewicz, J. P., and Kellogg, C. A. 2008. APPL ENVIRON MICROB, 74.24: 7828-7831. Toaza, A. et al. 2021. Plant Dis. Doi:10.1094/PDIS-02-21-0298-PDN Yamamoto, S., and Harayama, S. 1995. APPL ENVIRON MICROB, 61:1104.L. Sauer, A. V. et al. 2015. Agronomy Science and Biotechnology. Doi:10.33158/ASB.2015v1i1p21 Yu et al. 2021. Plant Dis. Doi:10.1094/PDIS-05-21-0988-PDN.
玉米（Zea mays L.）是中国最重要的农作物之一。自2020年以来，中国西南部的云南、四川和湖北等地出现了一种新的叶斑病。典型症状在抽雄后出现。病斑散生于叶片表面，圆形至椭圆形，直径3-20毫米。病斑初期呈水渍状，逐渐变黄或变白。2021年，在云南省曲靖市采集了具有典型白斑的病叶样本用于病原菌分离和鉴定。从每株植物坏死病斑边缘切取4小块叶片组织（面积约0.25平方厘米），用75%乙醇表面消毒1分钟，用无菌蒸馏水冲洗3次，然后在无菌蒸馏水中浸泡5分钟。将该溶液接种到Luria肉汤培养基（LB）平板上（Shin等人，2022年）。在28°C下培养24小时后，LB平板上出现了圆形、边缘光滑的黄色菌落。分离出的细菌为革兰氏阴性，氧化酶阴性，过氧化物酶、吲哚、柠檬酸盐阳性（Wells等人，1987年）。三株菌株（PA21QJ01、PA21QJ02和PA21QJ03）表现出相同的菌落形态。PA21QJ01用于进一步的分子分析。从在LB中培养的新鲜菌落中提取DNA（Shin等人，2022年），并分别使用引物27F/1492R（Galkiewicz和Kellogg，2008年）、UP-1/UP-2r（Yamamoto和Harayama，1995年）以及rpoBCM81-F/rpoBCM32b-R（Brady等人，2008年）扩增16S rDNA、gyrB和rpoB基因座的片段。分离株PA21QJ01的16S rDNA、gyrB和rpoB片段序列已存入GenBank（登录号：OM184301.1、OM302500、OM302499）。使用BLAST搜索同源序列，结果显示16S rDNA、gyrB和rpoB与菠萝泛菌NN08200的序列分别具有99.9%、99.6%和99.8%的同一性（GenBank登录号：16S rDNA为MK415050.1，gyrB为CP035034.1，rpoB为CP035034.1.1）。上述分子结果表明，从玉米白斑中分离出的PA21QJ01是菠萝泛菌。对感病玉米品种五谷1790的抽雄植株进行了致病性测试。在30°C的LB培养基平板上培养12小时后，将菌液以1×108 CFU ml-1的浓度用于接种。接种7天后，植株叶片出现水渍状。10天后，出现带有褐色边缘的白斑。相比之下，对照植株保持健康且无症状。在接种的玉米植株中重新分离出了相同的菠萝泛菌，符合柯赫法则。在过去十年中，据报道菠萝泛菌在南非、墨西哥、波兰、阿根廷、巴西（Sauer等人，2015年）和厄瓜多尔（Toaza等人，2021年）引起玉米白斑病。它还导致了与其他作物的病害，如洋葱、水稻、菠萝、甜瓜和高粱等（Sauer等人，2015年）。在中国，它导致水稻叶枯病和叶条斑病（Yu等人，2021年）。这是中国首次报道由菠萝泛菌引起的玉米白斑病。然而，据我们所知，这是中国首次报道玉米白斑病。应注意筛选抗病资源和培育抗病杂交种。参考文献：Wells, J. M.等人，1987年。《国际系统细菌学杂志》37:136 - 143。Shin, G. Y.等人，2022年。《植物病害》。DOI: 10.1094/PDIS - 08 - 21 - 1810 - SC。Brady, C.等人，2008年。《系统与应用微生物学》31:447。Galkiewicz, J. P.和Kellogg, C. A.，2008年。《应用与环境微生物学》，74.24: 7828 - 7831。Toaza, A.等人，2021年。《植物病害》。DOI:10.1094/PDIS - 02 - 21 - 0298 - PDN。Yamamoto, S.和Harayama, S.，1995年。《应用与环境微生物学》，61:1104。L. Sauer, A. V.等人，2015年。《农学科学与生物技术》。DOI:10.33158/ASB.2015v1i1p21。Yu等人，2021年。《植物病害》。DOI:10.1094/PDIS - 05 - 21 - 0988 - PDN。
