Progress 09/15/05 to 09/14/08

Outputs
OUTPUTS: A survey conducted in more than 100 acres at nine locations in seven municipalities of the major avocado production areas determined that P. cinnamomi and P. palmivora were directly related to root rot in the avocado orchards. Phytopthora nicotianae, P. hevea and Phytophthora species were identified from root and stem cankers in Juana Diaz. Nurseries were also infected with P. cinnamomi. Avocado root rot is a complex of at least two Phytophthora species. The development of cultivars resistant to the disease will need to include the different Phytophthora species in the screening programs. Phytophthora cinnamomi, P. palmivora and Phytophthora sp. were used in pathogenicity assays involving several avocado cultivars. Phytophthora cinnamomi was highly pathogenic on cultivars Wilson Popenoe, Buttler, Donaldson, Semil 34, and Simmonds. Catalina, Dusa and Semil 44, Millin, Avila and Russel were less affected by P. cinnamomi. Phytophthora palmivora also caused Phytopthora root rot in Wilson Popenoe, Buttler, Donaldson, Fushia, Simmonds, Avila, Russel and Catalina, but disease severity was considerably less than that caused by P. cinnamomi. Phytophthora sp. caused severe PRR in Wilson Popenoe, and moderate PRR in Donaldson. Our results indicate that future efforts to select cultivars with resistance to Phytophthora root rot should include a combination of the different Phytophthora species. In Florida accessions at USDA-ARS, Miami, were supplemented with germplasm collected in Central America (Fairchild Tropical Garden) and Puerto Rico (USDA-ARS). Increase in the breeding block accessions of the West Indian race and P. schiedeana from tropical and subtropical Latin America with several new accessions from Central America was established. All are putatively West Indian x P. schiedeana hybrids or pure West Indian avocados. The accessions were propagated by using budwood from trees in lowland sub-tropical and tropical areas with a high flood frequency. The new accessions were called 'Darien', 'Lizano', 'Edwin' and 'C.V. Laurance'. We proposed to find a management strategy for avocado root rot with the use of cover crops which alleviate soil physical factors that increase vulnerability of the trees to Phytophthora spp. Avocado trees showed the beneficial effect of cover crops by an increase in tree vigor and in infiltration, and a reduction in soil bulk density. At Juana Diaz, with the use of Arachis pintoi and Arachis glabrata, soil bulk density and aggregate stability percentage in the treatment with A. glabrata was higher than in the control, thus indicating significant soil structure improvement. Water infiltration rate in A. pintoi plots was significantly higher than in other treatments, probably because of a root system that allows better water displacement throughout soil pores. Percentage of total soil Nitrogen under A. glabrata was significantly higher than that for the control, thus indicating an improvement in soil fertility with the use of Arachis. Research results were presented in two local conferences in Puerto Rico and at a field day directed to growers and the Industry. PARTICIPANTS: Dr. Consuelo Estevez de Jensen: University of Puerto Rico, Assistant Researcher at Juana Diaz, Agricultural Experiment Station. Principal interests are soilborne pathogens and integrated disease management emphasizing the use of resistant varieties, cropping systems, biological control and reduced use of fungicides. Planned and lead project activities, survey, field and greenhouse trials. Participation in evaluations, research results analysis and writing reports. Dr. Victor Snyder: University of Puerto Rico, Professor Department of Agronomy and Soils, Coordination of research related to soil factors in avocado root rot. Chairman of a graduate student. Dr. Randy Ploetz: University of Florida. Research leader for the screening of avocado cultivars. Evaluation of the efficacy of the breeding program. Dr. Aaron Palmateer: University of Florida. Collaborated in the identification of Phytophthora. Prof. Felix Roman. University of Puerto Rico, Department of Horticulture. Participated in the field trials planning at Isabela Experiment Station. Prof. Luis Sanchez. University of Puerto Rico, Collaboration in the preparation of the rootstocks and rootstock screening. Prof. Rafael Ramos: University of Puerto Rico, Experimental Station Corozal. Agronomy Department. Assisted in the research involving cover crops. Prepared the plant materials of Arachis and Mucuna and also was involved in the evaluations of the cover crop trials. Mrs. Helen Violi. Post-doctoral student evaluated cultivars, parents and progenies against Phytophthora. TARGET AUDIENCES: A field day was conducted directed to growers and Extension Educators. Three Presentations on yearly professional meetings Two presentations on the Regional Meetings A presentation during the TSTAR evaluations on September 2008 directed to the TSTAR committee PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
This project addresses the combination of different approaches to control avocado root rot in Puerto Rico. The pathogenicity studies showed that the combination of P. cinnamomi and P. palmivora causes severe root rot. It was also observed that when both pathogens were isolated in the same orchard, DS was greater than when only P. cinnamomi was recovered. The most relevant finding was that the two species identified, P. cinnamomi and P. palmivora, were directly related to the symptoms and that differences in virulence were observed as previously reported. The isolation of more than one Phytophthora species from the same root sample is of great importance, since many studies have focused on P. cinnamomi and resistance studies are conducted with individual isolates. In Florida the introduction of accessions Darien, Lizano, Edwin and Laurance will increase the diversity of parents to breeding blocks. Clonally propagated rootstock, ungrafted and grafted with Simmonds scions, were used in greenhouse and field trials. We evaluated two selections for fine root attractiveness to P. cinnamomi zoospores relative to the most tolerant commercial variety, the "Dusa". Fine root attractiveness was evaluated by placing a total of 20 roots from each rootstock in a water-bath with motile zoospores. The number of zoospores attracted to each root was recorded. Rootstocks inoculated with P. cinnamomi and treated with Alliete to halt PRR were evaluated for root regeneration. As found in this investigation, the use of vegetative covers improved most of the soil physical properties evaluated, increasing aggregate stability percentage, reducing bulk density, increasing moisture retention and infiltration rate, thus providing more favorable conditions for the avocado orchard. In addition, the vegetative covers used in this investigation are efficient in nitrogen fixation. Arachis pintoi has been found to obtain up to 64% of its nitrogen from the atmosphere. Senescence and decomposition of the aerial parts, roots and the nodules, as well as nutrients from plant tissues all make nitrogen available to the avocado tree and other plants. In addition, the decomposition of vegetative tissues provides organic matter to the soil. The A. pintoi and A. glabrata cover crops will be useful for preventing soil degradation and for controlling avocado root rot. One disadvantage is the slow development of these covers and possible competition for plant nutrients.

Publications

• Borrone, JW; Olano, CT; Kuhn, DN, Violi, HA Schnell R. 2008. Outcrossing in Florida avocados as measured using microsatellite markers. Journal of the American Society for Horticultural Science Vol: 133 I: 2: 255-261.
• Borrone, JW; Schnell, RJ; Violi, H.A. 2007. Seventy microsatellite markers from Persea americana Miller (avocado) expressed sequence tags. Molecular ecology notes. Vol: 7 I: 3: 439-444.
• Estevez de Jensen, C., and Violi, H. 2008. Phytophthora root rot in avocado in Puerto Rico. Proceedings Third International Phytophthora, Pythium and Related Genera.
• Estevez de Jensen, C., Palmateer., A., Snyder, V., Roman, F., Ramos, R., Sanchez, L., Morales, R. and Torres, B. 2006. Integrated Management of Phytophthora Root Rot in Avocado in Puerto Rico. Proc. Interamer. Soc. Trop. Hort. 50:92-96.
• Estevez de Jensen, C. 2006. TSTAR Progress Report. University of Puerto Rico, 18 p.
• Torres, B. E., Estevez de Jensen, C., Snyder, V. and Vasquez, M. 2008. Relationship between vegetative covers and soil physical properties of one mollisol on Phytophthora cinnamomi ocurrence in avocado Persea americana Mill in Puerto Rico. Proceedings Caribbean Food Crops Society, Miami, FL.
• Violi, H.A., Borrone, J,W. Ploetz, R., Schnell R. 2008. Development of Phytophthora root rot-resistant avocado rootstocks for the Caribbean Conference Information: 119th Annual Meeting of the Florida-State-Horticultural-Society, Tampa FL. Proceedings Vol: 41-44.

Progress 09/15/06 to 09/14/07

Outputs
The morphological identification of six different Phytophthora isolates was based on the following parameters observed on PARP and V8 media at 16-h fotoperiod: hypha type, shape and size of clamydospores, shape and size of sporangia, and production of sexual structures. Molecular characterization was conducted for Phytophthora cinnamomi and P. palmivora. This study determined the presence of P. cinnamomi, P. palmivora, P nicotianae, and Phytophthora spp. in avocado orchards in Puerto Rico. Severe infection in avocado trees was triggered by a Phytophthora complex. Co-inoculation of Phytophthora species causes severe root rot. Phytophthora diversity is dependent on the dry or wet season conditions prevalent in the orchards that probably favor different Phytophthora species. Phytophthora palmivora is more commonly isolated during the dry season. These findings are important since limited research is directed to pathogen succession and effect on disease. Inoculation of the different Phytophthora species in avocado cultivars Millin and Semil 34 resulted in significant differences in disease severity, foliar and root weights. Phytophthora cinnamomi isolates ISA-LP-1S1-05, JD-EEA-IS3 and SG-IS5 were evaluated for differences in growth rate in vitro and levels of pathogenicity in avocado cultivars Millin and Semil 34. Further work is in progress to asses the resistance of clonal materials (developed at the USDA and University of Florida) of avocado varieties with tolerance to P. cinnamomi populations in Florida. Chemical analyses (except total N content) and moisture release curves were finalized on all soil samples collected to date from avocado grower farms. Moisture release curves from Martex farm samples were variable and are in the process of analysis. Moisture release curves obtained on four out of five commercial nurseries growing media indicated very little water released in the 0 to 30-cm tension range. Since avocado seedlings are typically grown in plastic bags approximately 30 cm high, maximum drainage suction in saturated soil cannot exceed -30 cm, thus indicating likelihood of anaerobic conditions in recently irrigated growing media. During the current final year of this project, now that the avocado and cover crop systems at Isabela and Fortuna are well established, each treatment is being instrumented with thermal dissipation probes to measure soil temperature and water matric potential, with capacitance probes for monitoring soil volumetric water content, and with Figaro galvanic cells for measuring oxygen concentration. Sensor readings at two-hour time intervals are controlled and captured on a CR-1000 data logger.

Impacts
Isolates of Phytophthora cinnamomi with variable level of virulence will be used in future studies of resistance. This study has important implications for breeding and selection for Phytophthora cinnamomi resistance in avocadoes.

Publications

• Torres, B., Estevez de Jensen, C., Snyder, V., Vasquez, M., and Ramos, R., 2006. Relacion entre propiedades fisicas del suelo e incidencia de Phytophthora en aguacate. Sociedad Puertorriquena de Ciencias Agricolas (SOPCA), 10 de Noviembre 2006, Guayanilla, Puerto Rico.
• Torres, B., Snyder, V., Vasquez, M. and Estevez de Jensen, C. 2007. Instrumentacion para medir propiedades fisicas del suelo en rizosfera de aguacate. Sociedad Puertorriquena de Ciencias Agricolas (SOPCA), Catano, Puerto Rico.

Progress 09/15/05 to 09/14/06

Outputs
A survey on the occurrence of Phytophthora cinnamomi (Pc.) and other soilborne pathogens associated with avocado root rot in selected avocado nurseries and orchards in Puerto Rico was initiated in November 2005. Seven orchards and four nurseries in Isabela, Juana Diaz and Santa Isabel were visited. In each orchard soil and root samples were collected from five avocado trees with symptoms of Phytophthora root rot as well as five samples from asymptomatic trees. Root samples were taken from the tree drip line and were transferred on specific media PARP (corn meal agar amended with antibiotics) and acidified potato dextrose agar (APDA). Disease severity of individual trees was rated by visual rating of tree foliage on a scale of 0 to 5 where 0 = healthy and 5 = completely defoliated. Root rot disease severity was scored in nursery trees on a scale 1 to 5 where 1 = healthy root and 5 = 100 % blackened roots. Soil samples were collected in the orchards with an 18-mm sampling tube at 0 to 5, 5 to 10, and 10 to 20 cm depth. The survey covered approximately 50 acres at six locations: Los Pinos (4 yr), San Sebastian (2.5 yr), Santa Isabel (10 yr), Isabela (10 yr), Juana Diaz (10 and 19 yr ), Villalba (4 yr) and Corozal (2 yr). Avocado root rot symptoms in trees included small pale green leaves with brown tips, wilting of the upper tree leaves, small and scarce fruit that fall readily and branches that break easily. Belowground, larger roots were brown and rotten and feeder roots were scarce or absent. Asymptomatic trees had green leaves, good fruit size and larger roots apparently healthy. However, feeder roots often had brownish color at the tips. Disease severity based on foliage symptoms in the different orchards ranged from 0 to 5. Most orchards, excluding Corozal and Villalba, showed well developed symptoms of Phytophthora root rot. Isolations on PARP media resulted in Pc in samples from Isabela and Santa Isabel, Juana Diaz, Los Pinos and San Sebastian. Phytophthora palmivora was identified in Juana Diaz; Pythium spp. was isolated from root samples in Isabela and Juana Diaz. Fungi isolated from avocado roots included multinucleate Rhizoctonia, Fusarium spp. DAS ELISA generalized test for the genus Phytophthora (AGDIA, Inc.) on roots collected in Santa Isabel resulted positive as well as samples from the four nurseries. Field trials were established in Juana Diaz and Isabela to determine the effect of cover crops on ARR. Twenty-four Arachis pintoi and A. glabrata plants were transplanted separately in the avocado tree canopy. Four randomly distributed trees were selected for each cover crop and a control was left with grass ground cover. In the second experiment avocado Semil 34 grafted rootstocks were planted in a field with a history of avocado root rot. Arachis pintoi and A. glabrata were transplanted in the avocado tree canopy. Control was left with no cover crop. This trial was replicated in Isabela. Disease resistant cultivars Dade and Catalina were grafted on Gripina rootstocks. Rootstocks from both cultivars will be developed by clonal propagation for further screen for Phytophthora resistance.

Impacts
The survey determined that avocado plants from nurseries are already infected with Phytophthora cinnamomi. Further analysis from seed beds using the ELISA test also resulted positive for Phytophthora spp. Isolations on specific media proved that Phytopthora spp. was present in avocado roots from seedlings in seed beds. These findings are important because they show that proper sanitation measures are needed in avocado seed beds and nursery substrates used for avocado plants.

Publications

• Estevez de Jensen, C. 2006. Soilborne Pathogens, Etiology and Management. Proceedings Workshop of Tropical Fruit Crops, Miami, Florida.
• Estevez de Jensen, C., A. Palmateer, F. Roman, S. Snyder, B. Torres, and R. Morales. 2006. Integrated Management of Phytophthora Root Rot in Avocado in Puerto Rico. Interamerican Horticulture Society Annual Meeting, San Juan, PR.