Several species of the Botryosphaeriaceae family have been associated with branch canker, dieback, and stem end rot in avocado (Persea americana Mill.). In Chile, the incidence of diseases affecting the avocado tree increased from 2011 to 2016, which coincided with a severe drought that affected avocado production. Moreover, distant countries importing avocados from Chile also reported an increase of stem end rot of ripe avocados. Therefore, the aims of this study were to identify the pathogen species associated with branch canker, dieback, and stem end rot of avocado in Chile and to study their pathogenicity. This study was conducted between 2015 and 2016 in ‘Hass’ avocado orchards located in the main avocado-producing regions in Chile. A diverse collection of fungal species was recovered from both necrotic woody tissue and necrotic tissue on harvested ripe fruit. On the basis of morphology and phylogenetic analyses of the internal transcribed spacer region (ITS1-5.8S-ITS2) and the translation elongation factor 1-α (TEF1-α) gene, eight species in the Botryosphaeriaceae family were identified: Diplodia mutila, D. pseudoseriata, D. seriata, Dothiorella iberica, Lasiodiplodia theobromae, Neofusicoccum australe, N. nonquaesitum, and N. parvum. For each of these species, pathogenicity studies were conducted on 1-year-old healthy Hass avocado plants. All isolates produced brown gum exudate and caused necrosis in the vascular system 3 weeks after inoculation. N. nonquaesitum, N. parvum, and D. pseudoseriata were the most virulent species. Necrotic lesions and cavities with white mycelia near the peduncle union were observed on Hass avocado fruit inoculated postharvest. L. theobromae, N. australe, and N. parvum were significantly more virulent than the other tested species in the Botryosphaeriaceae family. This study identified and characterized the pathogenicity of Botryosphaeriaceae species in Chile, which will prove useful to future research on these pathogens directed at establishing effective control strategies in avocado.

Avocado (Persea americana) production in Chile has increased to more than 33,500 ha. Chilean avocadoes are sent to markets 15 to 45 days away by overseas transport to the United States, Europe, and Asia. Although apparently healthy avocadoes were harvested in 2009, a 10 to 14% incidence of stem end rot appeared after 15 days of cold storage. Symptoms appeared as small, irregular, brown lesions on the peel at the stem end. Lesions enlarged rapidly, became sunken and soft, eventually comprising the entire fruit as ripening progressed. A white mycelium often developed around the stem cavity. A dark brown necrosis of the pulp was observed that comprised a big part of the pulp as the fruits matured. Isolations were performed from ‘Hass’ avocadoes that developed stem end rot after fruits were kept in humid chambers for 15 days at 5°C plus 6 days at 20°C (n = 50) to simulate a transport period from Chile to U.S. markets or from diseased fruits (n = 50) kept for 15 days at 20°C. Fruits were surface disinfected for 60 s in 75% ethanol, and small pieces of tissue were excised from the margins of the pulp lesions and then plated onto potato dextrose agar (PDA) plus 1 ml/liter of Igepal CO-630 (Sigma-Aldrich, Atlanta, GA) (MPDA). Fungal colonies that developed on PDA were white and cottony, turning slightly yellow after 15 days. Black acervuli appeared after 15 days at 20°C. Conidia (n = 40) were fusiform, (22.2) 27.0 to 30.4 × (6.3) 7.0 to 9.8 μm with a length/width ratio of 3.4 ± 0.4. All isolates had five-celled conidia. Apical and basal cells were colorless, while the three median cells were dark brown. Conidia had one basal appendage (9.3 ± 3.3 μm) and two to four long apical appendages (34.5 ± 6.9 μm). On the basis of colony and conidia morphology, most of these isolates were initially identified as Pestalotiopsis clavispora (G.F. Atk) Steyaert, but other nonidentified species of Pestalotiopsis were also found (3). Identification was confirmed by amplifying and sequencing the internal transcribed spacer (ITS) region of rDNA using ITS1/ITS4 primers of P. clavispora isolate PALUC-12 (Accession No. HQ659767). A BLAST search of the NCBI database showed that isolate PALUC-12 had 100% homology with P. clavispora (No. EU342214.1). Pathogenicity tests were conducted on surface-disinfected (75% ethanol, 30 s) fruits by placing agar pieces (3 mm in diameter) from 7-day-old cultures and a 20-μl drop of 106 conidia/ml on wounded and unwounded stem cavities and equatorial area of five avocado fruits of ‘Hass’, per isolate tested, at the commercial maturity stage. Inoculated fruits were placed in moist chambers at 25°C for 10 days. Necrotic lesions resembling symptoms that occurred in storage fruits were observed on wounded fruits. No symptoms were observed on unwounded fruits inoculated in the equatorial zone. However, unwounded fruits inoculated in the stem cavity developed a slight necrosis probably because of undetectable wounds made at harvest. Koch's postulates were confirmed after the reisolation of P. clavispora and Pestalotiopsis spp. from diseased fruits. P. versicolor has been reported in South Africa (1), but to our knowledge, this is the first report of P. clavispora causing stem end rot of avocado. P. clavispora has been reported on blueberry in Chile (2). References: (1) J. M. Darvas and J. M. Kotzé. Phytophylactica 19:83, 1987. (2) J. G. Espinoza et al. Plant Dis. 92:1407, 2008. (3) E. F. Guba. Monograph of Pestalotia and Monochaetia. Harvard University Press, Cambridge, MA, 1961.

In autumn 2013, fruit of Japanese plum (Prunus salicina) cvs. Angelino and Black Kat developed an unusual brown and soft rot after 2 months in cold storage (0°C) on nearly 1% of the fruit. Fruit showed small, circular, light brown spots that eventually destroyed the entire fruit. Small sporodochia appeared on the fruit surface. Fruit was harvested from orchards located near San Francisco de Mostazal (33°59′ S, 70°41′ W), Chile. Small pieces of diseased tissue were selected from margins of lesions of surface disinfected (96% ethanol) fruit (n = 7) and placed on acidified potato dextrose agar (PDA) plates for 5 days at 20°C. Light brown colonies with even margins and concentric rings of spores were obtained. The conidia of five isolates were one-celled, hyaline, lemon-shaped, (min. 10.7) 14.9 ± 1.5 (max. 18.6) × (min. 8.1) 9.4 ± 0.8 (max. 10.8) μm (n = 30), and borne in branched monilioid chains. This fungus was identified as Monilinia fructicola (G. Winter) Honey (1). Identification was confirmed by amplifying and sequencing the ribosomal ITS1-5.8S-ITS2 region using ITS1 and ITS4 primers (3). BLAST analysis of Chilean plum isolates (GenBank Accession Nos. KF148610 and KF148611) were 99 to 100% identical to isolates of M. fructicola originating from the United States (DQ314727 and HQ846966, respectively) and 100% identical to the first Chilean isolate (JN001480) found in nectarines originating from California at the supermarkets in Santiago in June 2009. Koch's postulates were fulfilled by reproducing brown rot symptoms on mature wounded Japanese plums cv. Angelino (n = 8) inoculated with 10 μl of a conidial suspension (105 conidia/ml) or with a mycelium plug (5-mm diameter). After 2 days in humid chambers (>80% relative humidity) at 25°C, all inoculated fruit developed brown rot symptoms with necrotic lesion means of 15.8 and 21.5 mm in diameter in fruit inoculated with conidia and mycelium, respectively. Non-inoculated control fruit remained healthy. Re-isolations were performed on PDA and the presence of M. fructicola was morphologically confirmed in 100% of the symptomatic fruits. To our knowledge, this is the first report demonstrating the presence of M. fructicola causing brown rot in stored Japanese plums in Chile after its first interception in 2009 in Chile, suggesting that this pathogen has been established in the field. Currently, M. fructicola is a quarantine organism under official control, restricted to Prunus orchards between Santiago and Nancagua in central Chile (2). References: (1) EPPO. EPPO Bull. 39:337, 2009. (2) Servicio Agrícola y Ganadero, SAG, Ministerio de Agricultura, Gobierno de Chile. www.sag.cl , accessed 15 November 2013. (3) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, NY, 1990.

Of 10 single-spore isolates tested, four showed similar disease reactions unique to pathotype I, four revealed pathotype II reactions, and one isolate each behaved like pathotype III or pathotype IV. SSR fingerprinting of these isolates provided evidence for genetic diversity since SSR ArH05T was highly polymorphic and amplified five bands, including pathotypes III-and IV-specific bands, which need further investigation to discern if this locus has any role to play in the virulence. MAT-type analysis showed that seven isolates were MATl-l while the remaining three isolates were MATl-2. Only pathotype I showed the profile of MATI-2 and the other three pathotypes were MATI-1. Initially, a number of chickpea wild relatives were screened to identify sources of resistance to pathotype IV, but none of the accessions tested showed resistance. However, efforts are underway to combine minor and major gene(s) available in the breeding program in addition to a further search of the wild gene pools to control pathotype IV.

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