First Report of Colletotrichum godetiae Causing Anthracnose and Twig Blight on Persian Walnut in Hungary

Abstract

Persian walnut (Juglans regia L.) fruit with preharvest anthracnose symptoms, necrotic fruit stalks, and twigs with necrotic buds, and peaks were collected in a Hungarian orchard next to Nágocs, in September 2018. Disease incidence was approximately 15% on a Hungarian bred walnut cultivar ‘Milotai 10’. Similar symptoms were found on Persian walnut in other locations (eg. Milota, Érd, Sarród, and Kocs). Acervuli were observed on necrotic lesions on fruit, and twigs with pale orange conidial masses. Conidia were hyaline, unicellular, and fusiform. Morphometric measurements of conidia showed mean length ± SD × width ± SD = 15.9 ± 1.7 × 4.5 ± 0.4 μm, length/width ratio 1:0.3 (n=100). The fungus was isolaed from conidial masses on potato dextrose agar (PDA) medium amended with Chlorampenicol (25 mg/L). A total of 12 isolates were obtained as pure cultures by single-spore isolations and incubated at 23°C in dark for 10 days. The colonies were white to gray or grayish-orange on the upper side and with black spots on the reverse side. The isolates showed morphological characteristics of Colletotrichum acutatum in sensu lato (Jayawardena et al. 2016). Molecular analyses were conducted to identify the exact species. Internal transcribed spacer (ITS) region, actin (ACT), and calmodulin (CAL) partial genes were amplified by ITS1F/ITS4R, ACT512F/ACT783R and CAL1/CAL2 primers (White at al. 1990, Carbone and Kohn 1999, O’Donnell et al. 2000). The sequences of ITS region (GenBank Accession Nos: MK367398-99, MK367401-02) showed 100% identity with C. godetiae sequence. Based on ACT gene (GenBank Accession Nos: MK415991-92, MK415994-95) were 100% identity with the deposited C. godetiae type strains from walnut. The obtained sequences of CAL gene (GenBank Accession Nos: MK415998-99, MK416001-02) were same and showed 100% with other C. godetiae sequences from other host plants. The fungus was identified as Colletotrichum godetiae Neerg. Pathogenicity tests were accomplished in the field and under laboratory conditions (25°C on thermostat) on 10 green ‘Milotai 10’ walnut fruit, and 10 walnut twigs each. Tests were conducted on living trees, collected fruit, and two-year-old twigs by inserting mycelial agar plugs (5 mm in diameter) onto wounded pericarp tissues, which were then wrapped with wet cotton and parafilm. Wounded tissues on 5 fruit and 5 two-year-old twigs were treated with non-colonized PDA plugs as noninoculated controls. After 14 d necrotic lesions 9 to 17 mm in diameter developed on fruit on living trees. Lengths of 12 to 17 mm and width of 7 to 12 mm necrosis was measured on phloem of walnut twigs, and almost two times larger in cambium. No necrosis developed around control wounds. Koch's postulates were fulfilled with the reisolation of the pathogen from symptomatic tissues, isolates were identical morphologically and by sequence analysis of ITS region, ACT, and CAL partial genes to the original isolates. Damm et al. (2012) described two C. godetiae strains associated with walnut, one isolated in Austria and another one of unknown origin. An epidemic event of walnut anthracnose caused by Colletotrichum species mainly C. godetiae was reported in France (Da Lio et al. 2018). The pathogen was isolated from nuts, buds, insects, and stems. To our knowledge, this is the first report of anthracnose of walnut fruit caused by C. godetiae in Hungary. Anthracnose caused by C. godetiae, and previously reported C. fioriniae (Varjas et al. 2019) is becoming an increasing preharvest problem on Persian walnut in Hungary.

Structural and Dynamic Analysis of Leaf-Associated Fungal Community of Walnut Leaves Infected by Leaf Spot Disease Based Illumina High-Throughput Sequencing Technology

“…The Colletotrichum spp., among the top 30 genera in relative abundance in our results, has become one of the top 10 internationally recognized phytopathogenic groups (Dean et al 2012). The genus consists of more than 100 species, such as C. gloeosporioides, C. hanaui, and C. fioriniae, all of these species can cause foliar fungal diseases on walnut trees (Qiu et al 2010;An and Yang 2014;Zhu et al 2015;Varjas et al 2021). Walnut brown spot is a complex pathogenic disease caused mainly by Fusarium spp.…”

Morphology and phylogeny of ascomycetes associated with walnut trees (Juglans regia) in Sichuan province, China

“…For example, walnut twig blight was caused by various fungi, viz. Neofusicoccum parvum , Phomopsis juglandina , Colletotrichum godetiae , and unidentified species in Diaporthaceae and Botryosphaeriaceae ( Thomidis et al, 2011 ; Cheon et al, 2013 ; Sun, 2013 ; López-Moral et al, 2020 ; Varjas et al, 2021 ). In addition, J. regia was usually infected by a range of canker disease pathogens, which often cause serious losses ( Fan et al, 2015b ).…”

“…In addition, J. regia was usually infected by a range of canker disease pathogens, which often cause serious losses ( Fan et al, 2015b ). Similarly, walnut anthracnose pathogens were also diverse ( Wang et al, 2017 , 2018 , 2020 ; Varjas et al, 2021 ). There are 17 most common fungal diseases occurring on leaves, twigs, and fruits of walnut trees in China, including more than 70 species of fungi, which are mostly ascomycetes ( Zhang et al, 2010 ; Li et al, 2015 , 2017 ; Wang et al, 2017 ; Fan et al, 2018 , 2020 ; Liu et al, 2020 ; Yang et al, 2021a , b ).…”

Molecular Identification and Characterization of Fusarium Associated with Walnut Branch Blight Disease in China

“…China is the world’s greatest producer of walnuts [ 4 ], accounting for approximately 48% of global production [ 5 ]. Walnut, on the other hand, is commonly affected with a variety of diseases such as cankers, blights, anthracnose, dieback, and kernel decay [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. Walnut branch blight (disease incidence of 3%) is widely documented to be a rising concern for walnut farmers in Gansu, Xinjiang, Henan, and other Chinese provinces, hurting fruit quality and output [ 6 ].…”

“…At least 10 species of pathogens are known to cause diseases in walnut worldwide, including Colletotrichum godetiae [ 7 ], Botryosphaeriaceae and Diaporthaceae species [ 8 , 9 , 10 ], Cytospora atrocirrhata , Colletotrichum chrysosperma , Colletotrichum gigalocus , Colletotrichum nivea , Colletotrichum sacculus [ 11 , 12 ], Boeremia exigua [ 13 ], Fusarium solani [ 14 ], Fusarium incarnatum [ 15 ], and Fusarium semitectum [ 16 ]. However, Fusarium is rarely reported on walnut branch blight.…”