Stem-end rot of harvested avocado fruit commonly occurs wherever the crop is cultivated. Multiple fungal species have been described as causal agents. To determine the causal pathogens of stem-end rot in California, fungal isolations were conducted from symptomatic fruit, and fungi were identified by morphological and molecular techniques. In 2010 and 2011, a total of 177 isolates were recovered from 290 avocado fruit collected from seven orchards in one of the major avocado growing areas in Southern California. The majority of isolates was identified as Neofusicoccum luteum (65%), with the remainder either as Colletotrichum gloeosporioides (33%) or Phomopsis sp. (2%). In a pathogenicity test, N. luteum caused significantly (P < 0.05) more severe stem-end rot than either C. gloeosporioides or Phomopsis sp. No significant (P > 0.05) differences in stem-end rot severity were observed between inoculations with N. luteum isolated from fruit stem-end rot and N. luteum or N. parvum isolated from branch cankers. This confirms that stem-end rot of avocado can be initiated by fungi causing branch cankers. Although low humidity and rainfall during much of the growing and harvest seasons in California are considered unfavorable conditions for the development of avocado stem-end rot, the identification of the causal pathogens is of value when decays have to be managed during outbreaks, and it stresses the importance of managing branch cankers.
Members of the family Botryosphaeriaceae cause branch cankers and dieback on California avocado trees. More intensive pruning, a practice associated with high-density planting that is becoming more common in the California avocado industry, may increase the occurrence of branch canker. This study was undertaken to identify and characterize the Botryosphaeriaceae spp. involved in the branch canker disease complex in order to develop future management strategies. From 2008 to 2009, branch cankers were sampled from four or five trees from each of eight avocado groves in five California counties. Six Botryosphaeriaceae spp. were identified based on morphology as well as phylogenetic analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) and a partial sequence of the β-tubulin gene. These six species included Neofusicoccum australe, N. luteum, N. parvum, an unknown Neofusicoccum sp., Fusicoccum aesculi, and Dothiorella iberica. Members of the Botryosphaeriaceae were isolated from all avocado-growing regions sampled in California; however, incidence and distribution of species varied. This report is the first description of the isolation of D. iberica from avocado branch cankers in California.
Phytophthora ramorum S. Werres & A.W.A.M. de Cock is the causal agent of sudden oak death in California and Oregon forests and ramorum blight on a broad range of host species in wildlands and nurseries. It is thought to be an introduced pathogen and only three clonal lineages are known (3). The North American lineage (lineage NA1, mating type A2) is responsible for infections in California and Oregon forests. The European lineage (lineage EU1, predominantly A1) is responsible for infections in Europe, but has also been found in nurseries in Oregon and Washington. A third lineage (NA2) has only been isolated in a few instances from nurseries in Washington and California. In June 2006, P. ramorum was isolated from diseased Viburnum tinus, Osmanthus heterophyllus, and O. fragrans cultivars from a Humboldt County retail nursery in northern California. We genotyped isolates and placed them into clonal lineages using microsatellite markers developed for P. ramorum (3,4). Genomic DNA was extracted from mycelia with the FastDNA SPIN kit (Q-Biogene, Morgan, Irvine, CA). Primers used were PrMS6, Pr9C3, PrMS39, PrMS43a, PrMS43b, and PrMS45 (3) and 18, 64, and 82 (4). We sized fluorescently labeled amplicons using capillary electrophoresis (3100 Avant Genetic Analyzer, Applied Biosystems, Foster City, CA). Isolate genotypes were compared with control isolates of known clonal lineage, including BBA9/95 (EU1), Pr102 (NA1), and WSDA3765 (NA2). Three of four isolates belonged to genotype EU1. The fourth isolate, obtained from O. fragrans, belonged to genotype NA1. We repeated genotyping on independent genomic DNA extractions and obtained identical results. Two EU1 isolates and the single NA1 isolate were tested for mating type (1) and found to be of A1, A1, and A2 mating type, respectively. The coexistence of A1 and A2 mating types in the same retail nursery suggests the potential for sexual reproduction, as is the case in P. infestans where clonal and sexual populations exist (2), although to date, sexual reproduction in nature has not been documented in P. ramorum. The California retail nursery infestation highlights the risks associated with the unintentional transport of host nursery stock infested with P. ramorum. References: (1) C. M. Brasier and S. Kirk. Mycol. Res. 108:823, 2004. (2) N. J. Grünwald and W. G. Flier. Ann. Rev. Phytopathol. 43:171, 2005. (3) K. Ivors et al. Mol. Ecol. 15:1493, 2006. (4) S. Prospero et al. Mol. Ecol. 16:2958, 2007.
In 1953, branch cankers on California avocado (Persea americana Mill.) trees were attributed to a Botryosphaeria anamorph, Dothiorella gregaria (teleomorph B. ribis) (2), and the disease was known as Dothiorella canker. Since this time, it has been suggested that this fungus should probably be classified as Fusicoccum aesculi Corda (teleomorph B. dothidea) (3). To our knowledge, B. dothidea is the only reported Botryosphaeriaceae species causing Dothiorella canker on avocado in California. Between the summer of 2008 and the winter of 2009, five trees from each of eight avocado orchards in five counties (San Diego, Riverside, Ventura, Santa Barbara, and San Luis Obispo) were surveyed for Dothiorella canker symptoms to verify the associated Botryosphaeriaceae species. Typical Dothiorella canker symptoms observed included darkened and friable bark with a dried, white, powdery exudate. Underneath the bark, cankers were variable in shape and some penetrated into the heartwood. Small sections of tissue (0.5 cm2) were excised from two to four separate cankers per tree and placed onto potato dextrose agar amended with tetracycline (0.01%) (PDA-tet). The most frequently isolated fungi, based on general growth pattern, speed, and colony color, were in the Botryosphaeriaceae with the following percent recovery by county: Riverside–40 and 100% (site 1 and 2, respectively); San Diego–60% (site 3); Ventura–42 and 53% (site 4 and 5, respectively); Santa Barbara–33% (site 6); and San Luis Obispo–32 and 60% (site 7 and 8, respectively). Pycnidia of Botryosphaeriaceae species were also observed on old diseased avocado tree branches. Sequenced rDNA fragments (ITS1, 5.8S rDNA, ITS2, amplified with ITS4 and ITS5 primers) were compared with sequences deposited in GenBank. Four different Botryosphaeriaceae species were identified and included Neofusicoccum australe, B. dothidea, N. luteum, and N. parvum, with species nomenclature based on the work of Crous et al. (1). Pathogenicity tests were conducted in the greenhouse on 1-year-old avocado seedlings, cv. Hass, with one randomly chosen isolate from each of the Botryosphaeriaceae species noted above. Four replicate seedlings were stem-wound inoculated with a mycelial plug and covered with Parafilm. Sterile PDA plugs were applied to four seedlings as a control. Over a period of 3 to 6 months, seedlings were assessed for disease symptoms that included browning of leaf edges and shoot dieback. Mean vascular lesion lengths on stems were 64, 66, 64, and 18 mm for B. dothidea, N. parvum, N. luteum, and N. australe, respectively. Each fungal isolate was consistently reisolated from inoculated seedlings, thus fulfilling Koch's postulates. To our knowledge, this is the first report of N. australe, N. luteum, and N. parvum recovered from branch cankers on avocado in California. These results are significant because Botryosphaeriaceae canker pathogens are known to enter the host plant through fresh wounds (pruning, frost, and mechanical). With high-density planting becoming more common, which requires intensive pruning, the transmission rate of these pathogens could increase in California avocado groves. References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) F. F. Halma and G. A. Zentmyer. Calif. Avocado Soc. Yearb. 38:156, 1953. (3) W. F. T. Hartill and K. R. Everett. N. Z. J. Crop Hortic. Sci. 30:249, 2002.
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