First Report of Fusarium meridionale Causing Stalk Rot of Ryegrass in China.
First Report of Fusarium meridionale Causing Stalk Rot of Ryegrass in China.
复制标题
我国首次报道南方镰刀菌引起黑麦草茎腐病。
DOI：
10.1094/pdis-11-21-2482-pdn
复制
发表时间：
2022-01
期刊：
影响因子：
4.5
通讯作者：
Wang Jianhua
中科院分区：
文献类型：
--
作者：
Wang Haoyu;Feng Disen;Chen Lingqiao;Yang Junhua;Wang Xichun;Wang Jianhua
禾谷镰刀菌复合体成员是引起小麦和其他谷物赤霉病、苗枯病或茎腐病的主要病原。FGSC种群的种类组成和真菌毒素产生的研究主要集中在小麦、玉米和大麦等粮食作物上，而对牧草中存在的FGSC病原菌的鉴定知之甚少。为了解多年生黑麦草(Lolium Perenne)茎腐病的病原学，于2021年4月在四川省洪雅县(103.37313 E，29.90661 N)进行了牧草病害调查。在几个牧场上观察到，多年生黑麦草约10%的产量损失是由茎腐病造成的。受影响的植株茎的颜色为棕色到深棕色，看起来很潮湿。随着感染的继续或在高湿度的条件下，一些植物的茎也被夷为平地。采集了有茎腐烂或茎褐变症状的多年生黑麦草样品。将症状组织切成小段(约5 mm)，用3%次氯酸钠溶液表面灭菌2min，用无菌蒸馏水冲洗3次，风干，接种在马铃薯葡萄糖琼脂(PDA)上，在28℃黑暗中培养，3~5d后，收集带有红橙色菌丝的镰刀菌菌落，转移到新的PDA平板上进行进一步纯化，单孢子分离得到纯化的培养物。获得了4个统一的分离物，它们在PDA上的菌落与典型的FGSC菌落相似(Leslie和Summerell 2006；O‘Donnell等人)。2004年)。在28℃黑暗条件下，PDA上菌落的平均径向生长速率为8.5~11.0 mm/d。对Wang等人描述的Spezieller Nährstoffer琼脂(SNA)的分生孢子特性进行了研究。(2014)。大分生孢子镰刀形至近直，背线与腹线平行，具3~5隔膜，长20.65~55.22μm，平均39.16μm，宽2.38~6.93μm，平均4.42μm(n=200)。没有观察到小孢子。对分离的镰刀菌菌株对健康多年生黑麦草(长江1号)的致病性进行了测定。种植2个月的黑麦草植株，用无菌牙签在茎上打一个洞，然后注射20μL大分生孢子悬液，浓度为105个孢子/毫升。以水处理黑麦草茎段为对照。每个处理包括20株植物。接种后，在25℃、90%湿度的生长室中培养24 h，3天后伤口处的茎组织变成棕色，棕色区域延伸到上面和下面的区域。水处理的对照组没有观察到任何症状。同样，从感染的草茎中重新分离出相同的病原菌，但没有从对照中分离到。因此，分离到的镰刀菌属。是多年生黑麦草茎腐烂的原因，这是基于科赫假设的实现。对镰刀菌进行鉴定。在物种水平上，按照Wang等人的描述，从所有四个菌株中扩增并测序了部分翻译延伸因子1-α(TEF)基因序列。(2015)。获得的序列完全相同，并将分离株SC1的序列提交给GenBank(登录号：MZ964308)。在两个数据库中对TEF序列(607bp)进行BLASTn搜索，结果表明该序列与子午镰刀菌菌株DS27的序列具有100%的相似性(登录号：菌株NRRL28723(http://isolate.fusariumdb.org/).)。一个串联的四基因系统发育树(补充图)将SC1和子午裂殖吸虫的模式标本(NRRL28723、29010和28436)分解在一个100%自举支持的单系分支中，确认SC1菌株属于子午裂殖吸虫。最后，按照Desjardins和Proctor(2011)的描述，使用在28°C黑暗中保存两周的大米培养物来评估子午裂殖吸虫菌株的三氯乙烯产量。LC-MS/MS分析表明，该真菌在水稻培养上能产生新城疫病毒和4硝基病毒，平均浓度分别为1400.44和3144.10μg/kg。据我们所知，这是中国首次报道子午孢霉在多年生黑麦草上致病。需要进一步的研究来确定它的分布、侵袭性和三氯乙烯的生产。
Members of the Fusarium graminearum species complex (FGSC) are the main causing agents of head blight, seedling blight, or stalk rot in wheat and other cereals worldwide. Surveys on species composition and mycotoxin production of FGSC populations have mainly focused on food crops such as wheat, maize, and barley, but little is known about the identity of FGSC pathogens present in pasture grass. In April 2021, a survey of grass diseases in the Hongya County (29.90661 N; 103.37313 E) in Sichuan Province was conducted to understand the etiology of stalk rot in perennial ryegrass (Lolium perenne). It was observed in several pastures that about 10% of yield loss in perennial ryegrass was caused by stalk rot. Affected plant stalks were brown to dark brown in colour and appeared soggy. As infections continued or under conditions of high humidity, some plant stalks also became flattened. Perennial ryegrass samples with symptoms of stalk rot or browning of the stem were collected. Symptomatic tissues were cut into short segments (approximately 5 mm), surface-sterilized in 3% sodium hypochlorite solution for 2 min, rinsed three times with sterile distilled water, air dried, plated onto potato dextrose agar (PDA), and then incubated in the dark at 28 °C. After 3 to 5 days, Fusarium-like fungal colonies with reddish-orange mycelium were collected and transferred to new PDA plates for further purification, and the purified cultures were obtained by single spore isolation. Four uniform isolates were obtained and their colonies on PDA resembled typical FGSC colonies (Leslie and Summerell 2006; O'Donnell et al. 2004). Colonies had an average radial growth rate of 8.5 to 11.0 mm/day at 28 °C in the dark on PDA. Conidial characteristics were studied on Spezieller Nährstoffarmer agar (SNA) as described by Wang et al. (2014). Macroconidia were falcate to almost straight, usually with parallel dorsal and ventral lines, 3- to 5-septate, 20.65 to 55.22 μm in length (average 39.16 μm), and 2.38 to 6.93 μm in width (average 4.42 μm) (n = 200). No microconidia were observed. The pathogenicity of the isolated Fusarium strains was then tested on healthy perennial ryegrass (variety Changjiang 1). Ryegrass plants grown for 2 months were inoculated by punching a hole in the stem using a sterile toothpick, followed by an injection of 20 μL macroconidia suspension at a concentration of 105 spores/mL. Ryegrass stems treated with water served as the control. Twenty plants were included in each treatment. After inoculation, the plants were grown in a growth chamber at 25 °C and 90% humidity for 24 h. Stalk tissues at the wound site turned brown after 3 days and the brown area then extended to regions above and below. No symptoms were observed in the water-treated controls. As well, the same pathogen was reisolated from the infected grass stems, but not from the controls. Thus, the isolated Fusarium spp. are a cause of stalk rot in perennial ryegrass based on the fulfillment of Koch's postulates. To identify the Fusarium spp. to species level, portions of the translation elongation factor 1-α (TEF) gene sequences from all four strains were amplified and sequenced as described by Wang et al. (2015). The obtained sequences were identical, and a sequence of isolate SC1 was submitted to GenBank (accession no. MZ964308). BLASTn searches were conducted on the TEF sequence (607 bp) in two databases, revealing it had 100% similarity to the sequence of Fusarium meridionale strain DS27 (accession no. MN629330) in NCBI and strain NRRL28723 from FUSARIUM-ID (http://isolate.fusariumdb.org/). A concatenated four-gene phylogeny (supplementary figure) resolved SC1 and the type specimens of F. meridionale (NRRL28723, 29010, and 28436) in a monophyletic clade with 100% bootstrap support, confirming that the strain SC1 belongs to F. meridionale. Finally, trichothecene productions of F. meridionale strains were evaluated using rice cultures kept at 28 °C in the dark for two weeks, as described by Desjardins and Proctor (2011). LC-MS/MS analysis indicated that the fungus could produce NIV and 4ANIV in rice cultures with average concentrations of 1400.44 and 3144.10 μg/kg, respectively. To the best of our knowledge, this is the first report of F. meridionale causing disease in perennial ryegrass in China. Further research will be necessary to determine its distribution, aggressiveness, and trichothecene production.