Lazreg F, Belabid L, Sanchez J, Gallego E, Garrido-Cardenas J A, Elhaitoum A
Dept. Agronomy, LRSBG, University of Mascara, PO Box 305, 29000 Mascara, Algeria.
Dept. Biology and Geology, University of Almeria, E-04120 Almeria, Spain.
Plant Dis. 2013 Apr;97(4):557. doi: 10.1094/PDIS-06-12-0608-PDN.
The Aleppo pine (Pinus halepensis Mill.) is a conifer native to the Mediterranean region. In autumn and spring of 2008 to 2009, a survey of Aleppo pine seedling diseases was carried out in three forest nurseries from the Relizane, Sidi Bel Abbes, and Tlemcen departments in northwestern Algeria. Aleppo pine seedlings were potted from the soil. In all three nurseries, 1- to 2-month old seedlings showed symptoms of damping-off disease in pre- and post-emergence (collar rot) with a disease incidence of 64, 77, and 72%, respectively. Disinfected collar segments, about 5 mm in length, were plated on PDA and petri dishes incubated at 25°C. A Fusarium sp. was consistently isolated from tissues and all isolates were morphologically identified as Fusarium acuminatum Ellis & Everh. (teleomorph: Gibberella acuminata Wollenw.) according to Fusarium keys (2). Colony growth was 43 mm after 3 days on PDA; the aerial mycelium was white, developing a brownish tinge in the center on PDA; macroconidia were formed in orange sporodochia, broadly falcate, strongly septate, 3 to 5 septa, the apical cell with an incurved elongation, distinct foot shape, 3 to 4 × 20 to 50 μm; microconidia were usually absent for isolates other than F12SS1, reniform, septate, 5 to 6 × 6 to 10 μm, in monophialides; chlamydospores were formed in chains, 6 to 13 μm. For the molecular identification, ITS regions of Fusarium isolates were amplified with the primers ITS1 and ITS4, and products were directly sequenced in both strands using the same primers ITS 1 and ITS4. Sequences were compared to known sequences deposited in the NCBI non redundant database to confirm morphological identification. An NCBI BLAST search identified isolates F12SS1, F14SS3, F30SS3, and F25SR as F. acuminatum based on 100% similarity with corresponding sequences. GenBank Accession Nos. were JX114788, JX114785, JX114782, and JX114790, respectively. Pathogenicity tests were performed to fulfill Koch's postulates. Inocula were produced by adding a 5-mm diameter plug from a 7-day-old CMA petri dish culture to a previously sterilized 500-ml flask (237.5 g sand, 12.5 g cornmeal, 80 ml SDW), shaken over 9 days, and mixed with sterile soil at 1:3 (v:v). The inocula were transferred to a 500-ml pot, and 10 Aleppo pine seeds were planted with three replicates. After 1 month, all tested isolates caused typical symptoms on seedlings and the proportion of infected seedlings per each isolate was 50, 53.33, 56.66, 60, and 63.33%, respectively. There are many reports of F. acuminatum associated to conifer seedlings in nurseries (1,3) and most of them are conflicting because in some reports this species is considered non-pathogenic or only a seed contaminant and others consider it as a pathogen. To our knowledge, F. acuminatum is a first report on the Aleppo pine in northwestern Algeria, northern Africa. It is also the first report of this fungal species affecting the Aleppo pine throughout the world, and on conifers in Africa and the Mediterranean region. References: (1) D. F. Farr and A. Y. Rossman. Fungal Databases, Systematic Mycology and Microbiology Laboratory. ARS, USDA., Bestville, Maryland, USA. Retrieved from http://nt.ars-grin.gov/fungaldatabases/ , June 18, 2012. (2) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, Iowa, USA, 2006. (3) D. W. Minter. Cybertruffle's Robigalia, Observations of Fungi and their Associated Organisms. Retrieved from http://www.cybertruffle.org.uk/robigalia/eng/ , June 18, 2012.
阿勒颇松（Pinus halepensis Mill.）是一种原产于地中海地区的针叶树。在2008年秋季至2009年春季，对阿尔及利亚西北部雷利赞、西迪贝勒阿巴斯和特莱姆森三省的三个森林苗圃中的阿勒颇松幼苗病害进行了调查。从土壤中挖出阿勒颇松幼苗进行盆栽。在所有三个苗圃中，1至2月龄的幼苗在出土前和出土后均表现出猝倒病症状（根颈腐烂），发病率分别为64%、77%和72%。将长度约为5毫米的消毒根颈段接种到PDA上，并将培养皿置于25°C下培养。始终从组织中分离出一种镰刀菌，根据镰刀菌分类检索表（2），所有分离株在形态上均鉴定为锐顶镰刀菌（Fusarium acuminatum Ellis & Everh.）（有性型：吉氏赤霉（Gibberella acuminata Wollenw.））。在PDA上培养3天后，菌落生长至43毫米；气生菌丝为白色，在PDA培养基中央逐渐变为褐色；大型分生孢子在橙色分生孢子座中形成，宽镰刀形，强烈分隔，具3至5个隔膜，顶端细胞弯曲伸长，呈明显的足形，大小为3至4×20至50微米；除F12SS1分离株外，其他分离株通常不产生小型分生孢子，小型分生孢子肾形，分隔，大小为5至6×6至10微米，在单瓶梗中产生；厚垣孢子成链状形成，大小为6至13微米。为进行分子鉴定，使用引物ITS1和ITS4扩增镰刀菌分离株的ITS区域，并使用相同引物ITS1和ITS4对产物的两条链进行直接测序。将序列与NCBI非冗余数据库中保存的已知序列进行比较，以确认形态学鉴定结果。通过NCBI BLAST搜索，基于与相应序列100%的相似性，鉴定分离株F12SS1、F14SS3、F30SS3和F25SR为锐顶镰刀菌。GenBank登录号分别为JX114788、JX114785、JX114782和JX114790。进行致病性测试以验证科赫法则。通过将来自7日龄CMA培养皿培养物的直径5毫米的菌块添加到先前灭菌的500毫升烧瓶（237.5克沙子、12.5克玉米粉、80毫升蒸馏水）中，振荡培养9天，并以1:3（体积比）与无菌土壤混合来制备接种物。将接种物转移到500毫升花盆中，种植10粒阿勒颇松种子，设置三个重复。1个月后，所有测试分离株均在幼苗上引起典型症状，每个分离株感染幼苗的比例分别为50%、53.33%、56.66%、60%和63.33%。有许多关于锐顶镰刀菌与苗圃中针叶树幼苗相关的报道（1,3），但大多数报道相互矛盾，因为在一些报道中该物种被认为是非致病性的或只是种子污染物，而其他报道则认为它是病原体。据我们所知，锐顶镰刀菌在北非阿尔及利亚西北部的阿勒颇松上的出现是首次报道。这也是该真菌物种在全世界范围内影响阿勒颇松以及在非洲和地中海地区影响针叶树的首次报道。参考文献：（1）D. F. Farr和A. Y. Rossman。真菌数据库，系统真菌学与微生物学实验室。美国农业部农业研究局，马里兰州贝茨维尔。检索自http://nt.ars-grin.gov/fungaldatabases/，2012年6月18日。（2）J. F. Leslie和B. A. Summerell。《镰刀菌实验室手册》。美国爱荷华州艾姆斯布莱克韦尔出版社，2006年。（3）D. W. Minter。Cybertruffle的Robigalia，真菌及其相关生物的观察。检索自http://www.cybertruffle.org.uk/robigalia/eng/，2012年6月18日。
