Biocontrol bacteria selected by a direct plant protection strategy against avocado white root rot show antagonism as a prevalent trait.

AIM: This study was undertaken to study bacterial strains obtained directly for their efficient direct control of the avocado white root rot, thus avoiding prescreening by any other possible mechanism of biocontrol which could bias the selection. METHODS AND RESULTS: A collection of 330 bacterial isolates was obtained from the roots and soil of healthy avocado trees. One hundred and forty-three representative bacterial isolates were tested in an avocado/Rosellinia test system, resulting in 22 presumptive protective strains, all of them identified mainly as Pseudomonas and Bacillus species. These 22 candidate strains were screened in a more accurate biocontrol trial, confirming protection of some strains (4 out of the 22). Analyses of the potential bacterial traits involved in the biocontrol activity suggest that different traits could act jointly in the final biocontrol response, but any of these traits were neither sufficient nor generalized for all the active bacteria. All the protective strains selected were antagonistic against some fungal root pathogens. CONCLUSIONS: Diverse bacteria with biocontrol activity could be obtained by a direct plant protection strategy of selection. All the biocontrol strains finally selected in this work were antagonistic, showing that antagonism is a prevalent trait in the biocontrol bacteria selected by a direct plant protection strategy. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the isolation of biocontrol bacterial strains using direct plant protection strategy in the system avocado/Rosellinia. Characterization of selected biocontrol bacterial strains obtained by a direct plant protection strategy showed that antagonism is a prevalent trait in the selected strains in this experimental system. This suggests that antagonism could be used as useful strategy to select biocontrol strains.

Avocado Dieback Caused by Neofusicoccum parvum in the Andalucia Region, Spain.

From 2002 to 2006, adult avocado trees, Persea americana Miller cv. Hass, located in the subtropical-fruit-producing area of Andalucia (southern Spain) developed symptoms of dieback characterized by death of twigs and branches in the tree canopy. Sections of surface-disinfested, necrotic branch tissues were plated on Difco potato dextrose agar (PDA) (Sparks, NV) and a Neofusicoccum-like fungus was isolated. On PDA, the isolates had white, appressed mycelium that turned dull gray as the colony aged, although conidia were not formed. Abundant pycnidia and conidia developed when isolates were cultured on 2% water agar with sterilized pine needles as substratum at 25°C under near-UV light for 2 weeks. Conidia were hyaline, unicellular, ellipsoid with an obtuse apex and subtruncate base, averaged 16.2 µm long by 5.8 µm wide and ranged from 12.0 to 20.0 by 4.0 to 8.0 µm, and becoming brown with one or two septa with age. Sequenced rDNA fragments (ITS1, 5.8S rDNA, and ITS2, amplified with ITS1 and ITS4 primers) of two avocado isolates were 100% homologous with Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers, & A.J.L. Phillips (1) (GenBank Accession Nos. AM410965 and AM410966). Morphological and molecular results confirmed this species as N. parvum, reported as the anamorph of Botryosphaeria parva (1). A pathogenicity test was conducted using two isolates on sets of five 2-year-old avocado plants produced from seeds of cv. Topa-Topa growing in 5-liter pots with soil. Unwounded and wounded plants were used for inoculations. Plants were wounded 2 to 3 cm below the apical tip with a lance (4 mm long and 1 mm deep). For inoculation, 4-mm 2-week-old PDA culture plugs were placed in contact with wounded tissues and covered with Parafilm. Five noninoculated plants treated similarly served as controls. Plants were maintained in the greenhouse with a temperature range of 18 to 26°C, and 1 month later, brown stem lesions, as much as 5 cm, originating from the inoculation site followed by dieback of branches were observed. Reisolations from necrotic branches were successful, and both isolates with identical morphology to those used for inoculations were recovered. Pathogenicity tests of seedlings using the same methods also caused stem lesions on unwounded plants and the pathogen was reisolated. To our knowledge, this is the first report of N. parvum causing dieback of avocado trees in Spain. Previously, B. parva has been reported causing stem-end rot of avocado fruit in New Zealand (2). In Spain, since diseased orchards are increasing rapidly, this pathogen could be efficiently distributed by pruning activities (tools and vegetal debris) as observed with other diseases (3). The presence of N. parvum in this subtropical area presents a serious disease problem not only to avocado but also to mango (Mangifera indica L.), which is another susceptible host (4). References: (1) P. W. Crous et al. Stud. Mycol. 55:235, 2006. (2) W. F. T. Hartill et al. N. Z. J. Crop Hortic. Sci. 30:249. 2002. (3) A. J. L. Phillips. Phytopathol. Mediterr. 41:3, 2002. (4) B. Slippers et al. Mycologia 97:99, 2005.

First Report of Phytophthora cactorum Causing Fruit Rot on Avocado in Spain.

The area of avocado (Persea americana Mill.) orchards in southern Spain has increased recently and is currently at 8,063 ha. Avocado production in this part of Spain was 72,581 t during 2003. During February 2004, apical necrosis was observed on avocado fruits (cv. Hass) in one orchard in Vélez-Málaga, Málaga Province, southern Spain. Dark brown lesions and necrotic flecking of the flesh also were observed on fruits. Isolations from the skin of the fruit previously washed with tap water and disinfested with 20% sodium hypochlorite on potato dextrose agar (PDA) consistently resulted in mycelial colonies. Sporangia produced on V8 juice by successive washing of mycelia with saline solution (1) measured 31 to 37.2 (33.3) × 21.7 to 28.8 (24.2) µm in size. The pathogen was identified as Phytophthora cactorum on the basis of morphological structures (mycelia, sporangia, chlamydospores, and oospores) formed when grown on V8 juice and PDA (2). To confirm pathogenicity, a mycelial suspension was obtained by blending mycelia grown for 1 week on PDA in 200 ml of sterile water. Three healthy avocado fruits were inoculated with the suspension by injection; three other fruits were inoculated by placing a drop of suspension on the unbroken skin of the fruit. The same number of fruit was inoculated as controls using sterile water instead of mycelial suspension. The inoculated fruits were incubated for 5 days in a moist chamber at 24°C in darkness. Spots appeared on all fruits for both inoculation methods, and the pathogen was isolated and identified as P. cactorum. No symptoms appeared on the control fruits. To our knowledge, this is the first report of P. cactorum causing fruit rot on avocado in Spain. References: (1) D. Chen and G. A. Zentmeyer. Mycologia 62:397, 1970. (2) G. M. Waterhouse and J. M. Waterston. No. 111 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, Surrey, UK, 1966.

Soil Solarization in Established Avocado Trees for Control of Dematophora necatrix.

Four field experiments on the control of Dematophora necatrix in avocado orchards affected by white root rot were conducted in the Mediterranean coastal area of southern Spain during 1991 to 1994. In the unshaded locations of solarized plots, the maximal temperatures were 35 to 42°C, depending upon the year and soil depth (15 to 60 cm). Temperature increases attributable to soil solarization ranged between 4 and 8°C in unshaded areas, whereas for shaded areas they were approximately 4°C. Inoculum recovery was decreased in root samples buried at 15 to 30 cm in unshaded locations of both solarized and unsolarized plots after 3 to 5 weeks, whereas 4 to 8 weeks of solarization were required for the elimination of the pathogen buried at depths of 45 to 60 cm. In contrast, inoculum recovery ranged from 30 to 60% for samples in shaded locations of unsolarized plots. D. necatrix was not recovered from roots of infected trees in solarized plots sampled 9 months after solarization, whereas recovery from roots in unsolarized plots was similar to levels before solarization. Soil solarization in established orchards was successful in reducing viability of inoculum buried in soil and eliminated inoculum in infected roots of live trees.