Morphological variability of Fusarium oxysporum f.sp.capsici isolates infecting pepper (Capsicum annuum L.) landraces in West Gojjam Zone, Ethiopia

The study was conducted in Jabi Tehinan, Burie Zuria, Dembecha Zuria and Womberma districts of West Gojjam Zone, Amhara Regional State, Ethiopia to characterize the morphological variability of Fusarium oxysporum f.sp.capsici isolates collected from the main pepper producing areas of Amhara region, West Gojjam, Ethiopia. These areas are known for high pepper production and incidence of Fusarium wilt. The study areas are situated in the Northwest direction of Addis Ababa at 385 km distance and 171 km South direction from the capital city of Amhara Region, Bahir Dar (Motbainor et al., 2016). The sampling areas are located between the geographical coordinates of 10°24’49” to 10°41’53” north latitude and 36°51’20” to 37°20’53” east longitude, with an altitude range of 1799 to 2115 m a.s.l. The districts have an average annual rainfall of 1168 mm and the mean annual temperature is 24.5°C. The sample collection districts were purposively selected based on their pepper production levels and expansion potential. From each district, four farmer associations were selected and from each farmer association, three representative farmers’ fields were selected purposively based on their scale of production and incidence of disease. In each affected field, plant samples were collected randomly, from root, stem and leaf parts (Abebe, A. & Abera, 2019). The sample plant parts were collected by looking into typical wilting symptoms including leaf yellowing, dropping, and partial or complete plant wilting (Akbar et al., 2018). Based on field size, 3–5 samples were collected per field (Altinok et al., 2019). Totally from all districts, 196 samples were collected and kept separately in labeled paper bags. The collected samples were placed in an icebox and taken to Injibara University, College of Natural and Computational Sciences, Microbiology and Parasitology Laboratory for identification. The samples were preserved at 4°C until isolation in the laboratory (Akbar et al., 2018; Endriyas et al., 2020). The sample collection was performed between September and October 2021 The collected symptomatic leaves, stems, and roots were thoroughly washed with running tap water. The washed parts were cut into small pieces (5 mm long) (Endriyas et al., 2020) and surface sterilized by soaking in 70% ethanol and 0.5% sodium hypochlorite solution (NaOCl) for 30 seconds and 1 minute, respectively (Oljira & Berta, 2020). The surface sterilized samples were rinsed for 1 minute in three successive changes of sterile distilled water and dried on filter paper (Altinok et al., 2019; Biri & Gomathinayagam, 2021). Then, the samples were placed on potato dextrose agar (PDA) plates (9 cm diameter) supplemented with chloramphenicol (0.25g/l) to restrict bacterial growth (Demissie et al., 2021; Kebede et al., 2020). All plates were labeled, sealed with parafilm and incubated at 25°C for 7 days to allow the development of mycelial growth of Fusarium (Getaneh et al., 2021; Hafizi et al., 2013). When the mycelia emerged, the colonies were sub-cultured onto a fresh sterile PDA to obtain a pure culture. Sub culturing was made till a distinct/pure culture are obtained and a pure culture of each isolate was stored on PDA slants at 4°C for further work (Hami et al., 2021). For the macroscopic data, colony color, pigmentation, margin, radial growth, shape, and mycelial growth pattern were recorded from the 7th day of pure culture following standard manuals (Campbell & Johnson, 2013; Leslie & Summerell, 2008; Nelson et al., 1983). Microscopic data were obtained using a compound microscope (400x) (Biri & Gomathinayagam, 2021) by preparing specimens from the 21 days grown mycelium.. Generally, morphological identification was performed according to the cultural characteristics and microscopic examination using the standard manuals used by different authors (Campbell & Johnson, 2013; Leslie & Summerell, 2008; Nelson et al., 1983). The raw data is indicated in the tables below.

本研究于埃塞俄比亚阿姆哈拉州西戈贾姆区的贾比·特希南、布里埃·祖里亚、登贝查·祖里亚及翁贝尔马地区开展，旨在表征从埃塞俄比亚阿姆哈拉州西戈贾姆辣椒主产区采集的尖孢镰刀菌辣椒专化型（Fusarium oxysporum f.sp.capsici）分离株的形态变异特征。这些地区以辣椒高产及枯萎病高发著称。研究区域位于亚的斯亚贝巴西北方向385公里处，距阿姆哈拉州首府巴赫达尔以南171公里（Motbainor等，2016）。采样区域地理坐标介于北纬10°24′49″至10°41′53″、东经36°51′20″至37°20′53″之间，海拔范围为1799至2115米。这些地区年均降雨量为1168毫米，年均气温为24.5℃。

样本采集区根据辣椒产量水平及扩张潜力进行目的性选择。每个地区选取4个农民协会，每个协会内根据生产规模及病害发生率目的性选取3块代表性农田。在每块受侵染农田中，从根、茎、叶部位随机采集植株样本（Abebe, A. & Abera, 2019）。样本采集基于典型枯萎症状，包括叶片黄化、脱落及植株部分或完全枯萎（Akbar等，2018）。根据农田面积，每块田采集3-5份样本（Altinok等，2019）。所有地区共采集196份样本，分别存放于带标签的纸袋中。

采集的样本置于冰盒内，送至因吉巴拉大学自然与计算科学学院微生物与寄生虫学实验室进行鉴定。样本在4℃下保存至实验室分离操作（Akbar等，2018；Endriyas等，2020）。样本采集于2021年9月至10月间完成。

带症状的叶片、茎和根经流水彻底冲洗后，切成5毫米长的小段（Endriyas等，2020），并通过依次浸泡于70%乙醇（30秒）和0.5%次氯酸钠溶液（NaOCl，1分钟）进行表面消毒（Oljira & Berta，2020）。表面消毒后的样本用无菌蒸馏水连续冲洗3次（每次1分钟），并在滤纸上干燥（Altinok等，2019；Biri & Gomathinayagam，2021）。随后将样本置于添加氯霉素（0.25g/L）的马铃薯葡萄糖琼脂（PDA）平板（直径9cm）上，以抑制细菌生长（Demissie等，2021；Kebede等，2020）。所有平板标记后用封口膜密封，于25℃培养7天，以促进镰刀菌菌丝生长（Getaneh等，2021；Hafizi等，2013）。菌丝出现后，将菌落继代培养至新鲜无菌PDA平板以获得纯培养物。重复继代培养直至获得单一纯培养物，各分离株的纯培养物保存于4℃的PDA斜面中以备后续研究（Hami等，2021）。

宏观数据方面，从纯培养第7天起，依据标准手册记录菌落颜色、色素沉着、边缘特征、径向生长、形态及菌丝生长模式（Campbell & Johnson，2013；Leslie & Summerell，2008；Nelson等，1983）。微观数据通过复式显微镜（400倍）获取（Biri & Gomathinayagam，2021），标本制备自培养21天的菌丝体。总体而言，形态鉴定基于培养特征及显微观察，采用不同作者使用的标准手册（Campbell & Johnson，2013；Leslie & Summerell，2008；Nelson等，1983）。原始数据详见下表。