CRB Funded Research Reports 2010 Research Project Progress Report Multiple Botryosphaeria species causing ‘Dothiorella’ gummosis in citrus Anthony Adesemoye, Akif Eskalen, Ben Faber and Neil O’Connell he occurrence of branch or trunk cankers on citrus counties in California, namely Fresno, Riverside, San Diego, caused by members of the Botryosphaeriaceae is San Luis Obispo (SLO), Tulare and Ventura. The branches Tknown as Dothiorella gummosis due to the gum or sap were transported on ice to the laboratory at the University that may exude from the canker. Symptoms include grayish of California Riverside (UCR). to brown discoloration on the bark, which could extend into Small pieces of cankered woody tissue were plated onto the xylem (Fig. 1). growth media, and different isolates of Botryosphaeria were Though Dothiorella gummosis is viewed as a minor disease obtained from them. Isolates were identified using morphol- in citrus, in advanced stages it can lead to serious decline or ogy and molecular methods. The molecular methods included death of branches, and in the case of young trees cause plant the use of three different molecular markers (Internal Tran- death. In a variety of woody hosts, many members of the scribed Spacer [ITS], Beta Tubulin, and Translation Elongation Botryosphaeriaceae are known to cause branch and trunk Factor). This was followed by sequencing, which was done at cankers and occasionally contribute to stem-end rot. the UCR Genomics Core facility. The sequences obtained Dothiorella gregaria (teleomorph: Botryosphaeria ribis) were then compared to the GenBank. was long believed to be the pathogen causing Dothiorella Teleomorph is a sexual reproductive stage, while ana- gummosis. However, isolation of Neofusicoccum vitifusiforme morph is an asexual reproductive stage of the same fungus. (Botryosphaeria sp.) from citrus branch canker by Eskalen and McDonald stimulated our hypothesis that there is more Pathogenicity tests were conducted for the strains by than one species of Botryosphaeria causing Dothiorella gum- inoculating green shoots. Fresh wounds were made on 1-year- mosis in citrus. old citrus shoots using a 3-mm cork borer, and the freshly Our objectives were to (a) determine the Botryosphaeria wounded surfaces were inoculated with mycelial plugs from species causing branch canker and dieback on citrus and their 3- to 5-day-old cultures of each fungus growing on potato geographical distribution in California, and (b) determine the dextrose agar amended with 0.01% tetracycline (PDA-tet), a pathogenicity and aggressiveness of the isolates. general medium used for culturing fungi. Control shoots were inoculated with sterile agar plugs. There were 10 replicates per How the study was conducted isolate and the control. In a survey of citrus orchards in California conducted in Inoculated wounds and shoot ends were covered with 2010, cankered branches were collected in six citrus growing petroleum jelly and wrapped with parafilm to prevent desic- Fig. 1. Samples of gummosis and branch canker caused by Botryosphaeria collected from three different locations. 32 Citrograph March/April 2011 (Left) Fig. 2. Sample pictures of 3-day-old fungal isolates growing on plates in the laboratory at UCR (UCP114 - Neofusicoccum mediterraneum; UCR1097 – N. luteum; UCR1099 – N. australe; and UCR1166 – N. parvum). 12 A A A 10 B B C BC (cm) 8 D DEF G DEFG DE GH FG FG G EFG H length 6 lesion 4 Shoot 2 I 0 UCP61 Control UCP105 UCP114 UCP124 UCP130 UCP134 UCP167 UCR1087 UCR1097 UCR1099 UCR1104 UCR1110 UCR1111 UCR1116 UCR1130 UCR1166 UCR1177 UCR1180 Fig. 3. Mean lesion length on shoots after test inoculation. Detail of names for listed strains can be seen in Table 1. Mean with different letters are significantly different at p=0.05. cation. Shoots were incubated at 25°C Table 1: Botryosphaeria isolates fully identified morphologically and genetically. in moist chambers for four weeks. Le- Strain.. Organisms. Teleomorph. County. Rootstock. Scion sions were observed on all inoculated uCP61 Dothiorella viticola Spencermartinsia viticola* Tulare Sour orange Valencia shoots except for the controls. The length of each lesion was recorded. uCP105 Dothiorella viticola Spencermartinsia viticola Tulare Sour orange Parent Washington Re-isolation from inoculated tissues was done to complete the pathogenic- uCP114 Neofusicoccum Unknown Tulare Carrizo Fukumoto mediterraneum ity of isolates. uCP124 Dothiorella iberica Botryosphaeria iberica Tulare Carrizo Fukumoto Findings and the implications uCP130 Diplodia mutila Botryosphaeria stevensii Tulare Carrizo Fukumoto As can be seen in Figure 2, some uCP134 Dothiorella viticola Spencermartinsia viticola Tulare Sour orange Parent of the species of Botryosphaeria are Washington very similar in morphology. Some are uCP167 Dothiorella viticola Spencermartinsia viticola Tulare Sour orange Valencia also very similar in genetic composi- uCR1087 Neofusicoccum Botryosphaeria sp. San Diego Sour orange Red blush tion. The combination of morphology mangiferae grapefruit with three types of molecular mark- uCR1097 Neofusicoccum luteum Botryosphaeria lutea San Diego Unknown Valencia ers helped to fully identify isolates uCR1099 Neofusicoccum australe Botryosphaeria australis San Diego Unknown Valencia and distinguish among closely related uCR1104 Dothiorella viticola Spencermartinsia viticola San Diego Unknown Valencia species. Hence, the 18 isolates listed in Table 1 have been fully identified. uCR1110 Neofusicoccum australe Botryosphaeria australis Riverside Unknown Satsuma The common form of the organism uCR1111 Neofusicoccum australe Botryosphaeria australis Riverside Unknown Satsuma seen in nature is the anamorph, but uCR1116 Neofusicoccum Botryosphaeria sp. Fresno Unknown Mandarin the teleomorph names are included mangiferae Nova on the table for better understanding. uCR1130 Dothiorellaviticola Spencermartinsia viticola Riverside Unknown Eureka Grower records were consulted to Lemon obtain information on the age of the uCR1166 Neofusicoccum parvum Botryosphaeria parva Ventura Volkameriana Meyer trees, rootstock and scion, irrigation Lemon management, cultural and fertiliza- uCR1177 Neofusicoccum luteum Botryosphaeria lutea SLO Unknown Lemon tion practices, and disease and pest uCR1180 Neofusicoccum luteum Botryosphaeria lutea SLO Unknown Lemon histories in the orchard. The informa- *Spencermartinsia viticola is also called botryosphaeria viticola (basionym). Basionym means the first or previous name that tion shows that isolates were collected was given to the organism. from both organic and non-organic farms. All ages of trees shoots in the laboratory (Figure 3), the isolates are pathogenic appear susceptible to the infection, as the hosts from which on citrus. All isolates were recovered 100% from lesions in isolations were made varied from 5 to over 60 years old. Com- each respective set of infected shoots. However, the study monly used rootstocks – ‘Carrizo’, Citrus volkameriana, and will continue with further tests in the greenhouse to better sour orange – were infected. understand their pathogenicity and aggressiveness. Based on the pathogenicity tests conducted using green Results indicate that multiple species of Botryospharia, March/April 2011 Citrograph 33 including B.australis, B. iberica, B. lutea, B. species, N. medi- is very important to maintain optimum tree health, free of terraneum, B. parva, B. stevensii, and S. viticola, were causing wounds, and ensure high levels of sanitation in the orchard. symptoms on citrus that were previously believed to be Also, controlling damage by gophers and meadow mice seems caused by Dothiorella gregaria (B. ribis). to be important as the wounds can be points of entry. It was observed that these multiple species were widely Notably, Dothiorella gregaria was not isolated from any distributed within counties. For example, in Tulare County of the counties. The term “Dothiorella gummosis” now ap- where seven isolates were obtained, four of them were S. viti- pears to be a misnomer, as anamorphs of many other species cola while three were different species (N. mediterraneum, B. of Botryosphaeria have been determined to cause the same iberica, and B. stevensii). In San Diego, four different species symptoms. It may be more appropriate to refer to the disease were isolated (B. lutea, B. australis, S. viticola, and B. species). as “Botryosphaeria gummosis”. To our knowledge, this is the Two species were identified in Riverside (B. australis, and S. first comprehensive study of Botryosphaeria gummosis on viticola) and one species each in Fresno (Botryosphaeria sp.), citrus in California. These findings are important and should Ventura (B. parva), and San Luis Obispo (B. lutea). be considered in the design of future studies on management The gumming, as seen in Figure 1B, was generally more techniques of Botryosphaeria gummosis in citrus. common in the summer and early fall but was hardly seen in Project leader Dr. Akif Eskalen is Cooperative Exten- winter and spring. It has previously been shown in other hosts sion Specialist and Plant Pathologist, and Anthony Ad- that both ascospores (sexual spores) and conidia (asexual esemoye is a postdoctoral scholar, Department of Plant spores) produced by Botryosphaeria in structures that can Pathology and Microbiology, University of California be found on dead bark, twigs and cankers are infective. The Riverside. Dr. Ben Faber is a UC Cooperative Extension spores enter the host plant through fresh wounds such as farm advisor