POWDERY MILDEWS: INVASIVE AND EMERGING DISEASES IN THE TROPICS.

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: SPECIAL GRANT
• Reporting Frequency: Annual
• Accession No.: 0219797
• Grant No.: 2009-34135-20136
• Proposal No.: 2009-04028
• Project Start Date: Sep 1, 2009
• Project End Date: Aug 31, 2012
• Grant Year: 2009
• Program Code: [AH]- Tropical & Subtropical Research/T STAR

Recipient Organization
UNIVERSITY OF PUERTO RICO AT MAYAGUEZ
P. O. BOX 9000
MAYAGUEZ,PR 00681

Performing Department
Crops and Agroenvironmental Sciences

Non Technical Summary
Situation: Knowledge of plant pathogenic fungi biodiversity is an important issue in Puerto Rico's fragile agricultural ecosystem and fundamental when dealing with exotic plant diseases of quarantine importance. Damaging fungal pathogens such as the powdery mildews are responsible for major epidemics through the history, and have been placed at the top list of plant diseases of economic importance worldwide. They are obligate parasites, easily recognized by their symptoms on the surface of plant tissues but limited knowledge of their taxonomy; misconceptions of their reproductive biology (i.e., usually considered host specific pathogens); and major phytosanitary issues based on their ability to become invasive across international borders, as they are usually not easily detected in cuttings or other propagative plant materials during quarantine inspections, make them an important group to study. Until recently, their taxonomy was based on teleomorphic characters or sexual stages, rarely found in the tropics thus making accurate identifications unfeasible. In fact, plant disease literature is characterized by hundreds of reports of powdery mildews identified only as Oidium sp. Recent taxonomic studies in Europe, Australia and Japan have focused in resolving the generic Oidium issue by using a combination of scanning electron microscopy and molecular biology tools (i.e. specific nucleotide sequences of the rDNA ITS region). Purpose: We propose to apply aforementioned techniques, in conjunction with host range data to establish a data base of correct identified anamorphic powdery mildews occurring in the island. This step is crucial not only when dealing with exotic plant diseases of quarantine importance but when breeding for resistance and when establishing effective integrated pest control programs.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	1099	1102	50%
212	1099	1040	50%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
1099 - Tropical/subtropical fruit, general/other;

Field Of Science
1040 - Molecular biology; 1102 - Mycology;

Keywords

powdery mildews

ascomycetes

erysiphales

Goals / Objectives
1. Conduct a survey of powdery mildews occurring in Puerto Rico and establish a database of identified specimens. 2. Identify anamorphic powdery mildews using Scanning Electron Microscopy and molecular biology tools (i.e rDNA ITS region). 3. Match anamorphic specimens to their teleomorph using DNA sequences from the GenBank.

Project Methods
Disease identification and plant material recollection: Freshly infected plant material showing symptoms that resemble powdery mildew will be collected in plastic bags and transferred to the Plant Pathology Laboratory at UPR-Mayaguez Campus or to the Plant Diagnostic Clinic located at the Agriculture Experiment Station in Juana Diaz, Puerto Rico. Photographic documentation of host plant and lesions will be taken to create a digital herbarium. Information of plant genera and species, date of recollection, collector's name, place of recollection, common name will be recorded and a specimen herbarium will be deposited. Microscopy: Light and SEM: Microscope slides will be prepared of longitudinal and transversal sections of plant tissue. Data of conidial size and shape, arrangement (chain or single), presence or absence of fibrosin bodies and germination pattern will be recorded. For Scanning Electron Microscopy (SEM), small sections (2-4 mm) of healthy and symptomatic tissue (1mm x 1mm) will be fixed in phosphate buffer with 4% glutaraldehyde. Samples will be dehydrated in a series of ethyl alcohol and subjected to critical point drying before their examination with the SEM (Model JEOL JSM-5410LV). For powdery mildew characterization using SEM, detail observations of patterns on the surface of the conidia, conidiogenesis, germination pattern, appresoria and type of parasitism will be recorded (Cook et al., 1997; Braun et al., 2002). Genomic DNA extraction and amplification of ITS region of the rDNA gene: Powdery mildew genomic DNA will be extracted using DNeasy Plant Mini Kit, Qiagen, CA. DNA samples will be stored at -20 Celsius degree for further use. Amplification by PCR of the ITS of the rDNA genes will be performed using specific primers: ITS1 and ITS5 using a thermocycler (Perkin Elmer Model 2400) (Cunnington et al., 2003; White et al., 1990). PCR conditions will be: initial DNA denaturizing for 10 min at 94 Celsius degree and 35 cycles of: 1min at 94 Celsius degree, 1min at 65 Celsius degree, 2 min at 72 C degree and a final extension of 10 min at 72 Celsius degree. A 1.4 % agarose gels with ethidium bromide will be used to visualize PCR products in a electrophoresis documentation system (BIO RAD). PCR products will be sequenced directly using the ITS4 and ITS5. DNA amplification for each fungal isolate will be repeated twice and samples will be stored at -20 Celsius for further use. DNA sequencing will be performed by commercially available services. The resulting sequences will be aligned by clustal method (www.ebi.ac.uk/clustalw). A dendogram will be constructed including DNA sequences obtained from the GeneBank data base. Phylogenetic trees will be created using the program Phylo_win(copyright) developed for Genome Populations, Interactions Laboratory from the Montpellier University in France. Sequences from other species of powdery mildews, especially teleomorphs will be obtained from the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/) for comparison and phylogenetic trees development.

Progress 09/01/09 to 08/31/12

Outputs
OUTPUTS: Activities: Survey: From 2010 to 2012, samples of powdery mildews (Ascomycetes: Order Erysiphales) affecting diverse plant species were collected in west, central and southern Puerto Rico. Related to Teaching and Mentoring: Throughout the course of the project, one undergraduate and four graduate students were trained in fungal morphological identification using light and Scanning Electron Microscopy (SEM), focusing on powdery mildew species. In addition, they were trained in the use of DNA-based techniques commonly used in fungal identification. One of them finished the M.S. degree in Crop Protection at UPR; another M.S. thesis is in progress. Events: During the scope of the project, findings were disseminated at regional, national and international plant pathology conferences such as the national meeting of the American Phytopathological Society (APS) joined with the International Association for Plant Protection Sciences held in Hawaii (2011), and at two APS-Caribbean Division meetings held in Nicaragua (2010) and in Puerto Rico (2011). Locally, findings were presented at the annual meeting of the Puerto Rican Mycological Society and at the Puerto Rican Society of Agricultural Sciences (2010 & 2011) held at different locations throughout the island. The PI presented these findings as plenary speaker invited to Mycology Symposium in 2010. Product: An informative web page was designed to provide open access online to the database pertaining to powdery mildew occurring in Puerto Rico (http://erysiphalespr.eea.uprm.edu/). PARTICIPANTS: Lydia I. Rivera-Vargas, PI: Project leader, in charge of the logistics of the project. Student advisor and research mentor, trained students in fungal morphological identification and in the application of DNA-based technologies. Consuelo Estevez de Jensen , Co-PI: Assisting PI in project logistics; student advisor, assisted in student training of fungal morphological identification and in the application of DNA-based technologies. Melissa Vaz, undergraduate student. She was trained in the identification of powdery mildew specimens, using DNA analysis. Carlos Bolanos, Laura L. Bonilla, Elena Latoni-Brailowsky and Luz M. Serrato, graduate students. They were trained in the identification of powdery mildew specimens, using light, SEM and DNA-based techniques. Collaborators: Carlos Munoz, Ph.D.; and Jose Almodovar, M.S., Biology Department, Microscopy Center, UPR-Mayaguez Campus. In charge of SEM facilities, assisting students in the preparation of samples and use of SEM. Professional development: All undergraduate and graduate students involved in the project attended regional, national and international meetings to offer oral or poster presentations of their findings. TARGET AUDIENCES: Undergraduate and graduate Hispanic students acquired skills in the identification of phytopathogenic fungi by using novel technologies. Hispanic ornamental and vegetable growers were visited during the scope of the project and thus became aware of powdery mildew infections. PROJECT MODIFICATIONS: Powdery mildews are obligate parasites that cannot be cultured in vitro. Obtaining good quality DNA sequences of powdery mildews was challenging. Two major unexpected outcomes should be mentioned: In the tropics, we observed only powdery mildew anamorphic phases, which are usually parasitized by other fungi such as Ampelomyces sp. The genetic region used for powdery mildew characterization (i.e. rDNA ITS region) is not specific. Even when using previously published specific powdery mildew primers, we were unable to obtain good quality DNA sequences to allow for species identification. In addition, on certain occasions plant DNA was also amplified by using rDNA ITS primers. This situation requires further investigation.

Impacts
Change in knowledge: Fourteen powdery mildew species were identified from 38 different plant species by using light and SEM, as well as DNA-based techniques. The most common genus was Erysiphe, with eleven different species affecting mainly ornamentals and weeds: E. acalyphae, E. australiana, E. begoniicola, E. heraclei, E, peruviana, E. polonica, E. quercicola, and E. russellii. Erysiphe trifolii was found affecting beans (Phaseolus vulgaris) and E. poligoni, lettuce (Lactuca sativa). In vegetables the most common among the genera was Podosphaera. Podosphaera xanthii was identified affecting various species of cucurbits: Cucumis melo, C. pepo and Citrullus lanatus. Podosphaera fusca was identified in okra (Abelmoschus esculentus). Erysiphe australiana, E. pisi, Podosphaera fusca and P. pannosa identification was confirmed by using DNA analysis of the rDNA ITS region. Twelve species are new reports of powdery mildews for Puerto Rico. These are: Erysiphe peruviana, E. polonica, E. quercicola, E. rusellii, E. trifolii, Oidium bixae, O. caricae, O. mangiferae, O. neolycopercisi, O. poinsettia, Podosphaera fusca, and P. xanthii Change in actions: Correct identification of powdery mildew is crucial not only when dealing with exotic plant diseases of quarantine importance but also when breeding for resistance and when establishing effective integrated pest control programs, especially in Puerto Rico's fragile agricultural ecosystem. Change in conditions: This is the first study focused on the identification of powdery mildews in Puerto Rico. The findings of this project will provide knowledge for a better disease management and crop quality.

Publications

• Abstracts: Bolanos, C., C. Estevez de Jensen and L.I. Rivera Vargas. 2010. Characterization and identification of powdery mildews from legumes and weeds in Puerto Rico. Proceedings of the Annual Meeting of the Puerto Rican Society of Agricultural Sciences (SOPCA), Hato Rey, P.R.
• Bonilla Aviles, L. L., C. Estevez, L.I. Rivera Vargas and B. Roman. 2011. Powdery mildews of ornamental plants in Puerto Rico. Proceedings of the Annual Meeting of the Puerto Rican Society of Agricultural Sciences (SOPCA), Ponce P.R.
• Bonilla, L.L., C. Estevez de Jensen and L.I. Rivera Vargas. 2010. Identification of Golovinomyces sp. in Acalypha wilkesiana using Scanning Electron Microscopy and other classification criteria. Phytopathology 101 (6):S253-254.
• Bonilla Aviles, L.L., C. Estevez de Jensen and L.I. Rivera Vargas. 2011. Identification of powdery mildews affecting ornamentals and herbs in Puerto Rico. Annual Meeting American Phytopathological Society-Caribbean Division, San Juan, Puerto Rico, Phytopathology 101(6):S276.
• Bonilla Aviles, L.L., L.M. Serrato Diaz and L.I. Rivera Vargas. 2010. Morphological characterization of fungi occurring in vegetables in Puerto Rico. XII Mycology Simposium, Interamerican University Aguadilla, P.R.
• Latoni-Brailowsky, E., M. Vaz and L.I. Rivera-Vargas. 2010. Identification of Oidium neolycopersici, a powdery mildew of tomato, using scanning electron microscopy (SEM) and DNA analysis. Phytopathology 101(6):S254.
• Rivera-Vargas, L.I. 2010. Tropical Phytopathogenic Fungi: Advances and Challenges. Plenary speaker at the XII Mycology Symposium, Mycological Puerto Rican Society held at Interamerican University, Aguadilla, P.R.
• Rivera-Vargas, L.I. 2011. Applications of PCR-Based techniques in the diagnostics of tropical plant pathogens. Phytopathology 101 (6): S255.
• Rivera L.I , E. Latoni and C. Estevez. 2011. Open access online database of powdery mildews (Order Erysiphales) in Puerto Rico. Phytopathology 101 (6):S154.
• M.S. Theses: Bonilla Aviles, Laura L. 2012. Identification of anamorphic species of powdery mildews occurring in ornamentals and aromatic herbs in Puerto Rico. M.S. Thesis. Department of Crops and Agro-Environmental Sciences. University of Puerto Rico-Mayaguez Campus. Mayaguez, P.R., 130 pp.
• Latoni Brailowsky, Elena. 2013. Identification of powdery mildews (Ascomycota; Orden Erysiphales) occurring in fruits and vegetables using Scanning Electron Microscopy and DNA analysis. M.S. Thesis. Dept. Crops and Agro-Environmental Sciences, University of Puerto Rico-Mayaguez Campus, Mayaguez, P.R. (in progress).

Progress 09/01/10 to 08/31/11

Outputs
OUTPUTS: Project findings have been disseminated at national and international Plant Pathology meetings such as the American Phytopathological Society, National and Caribbean Division annual Meetings held in Hawaii and Puerto Rico, respectively. Information was presented on the identification of powdery mildew species (Ascomycetes: Order Erysiphales) associated with fruits, ornamentals, vegetables and weeds in the tropics. This was relevant to the audience since more than 400 participants attended the Caribbean Division Meeting and more than 1,600 the national meeting. Very limited studies of powdery mildews of the tropics have here before been published. In ornamentals, the identification of Erysiphe sp. affecting bigleaf hydrangea (Hydrangea macrophylla) will be important for the implementation of management practices. Erysiphe peruviana was identified in Cape honeysuckle, Tecomaria capensis. These diseases were previously identified as caused by undetermined Oidium species. This work is an advance in species identification to prevent the importation in Puerto Rico of ornamentals which represent a pathway to new disease introduction. In addition, two different species of powdery mildew were identified in papaya: Oidium sp. and the common Oidium caricae. Podosphaera xanthii were found infecting various species of cucurbits on the island. For the first time Podosphaera fusca was identified causing powdery mildew of okra, Abelmoschus esculentus. Erysiphe heraclei, E. peruviana, E. quericola, Podosphaera fusca and P. xanthii are new reports for Puerto Rico. PARTICIPANTS: E. Latoni-Brailowsky and L. Bonilla: graduate students working in the identification of powdery mildew specimens. Professional development: Oral presentations were made by two graduate students, E. Latoni-Brailowsky and L. Bonilla, at the American Phytopathological Society - Caribbean Division (APS-CD) Annual Meeting joint with the Southeastern Branch of the Entomological Society of America held at San Juan, Puerto Rico. Poster presentation made at the American Phytopathological Society Annual Meeting joint with the International Association for the Plant Protection Sciences held at Honolulu, Hawaii. Project advances were presented. TARGET AUDIENCES: During the collection of ornamental samples, growers from the central area of Puerto Rico (Aibonito), an important municipality for the industry, were visited. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
From 2010 to 2011, samples were collected throughout the island of Puerto Rico. Forty- nine different plant species were examined for powdery mildew (Ascomycetes: Order Erysiphales) infection. Using light and scanning electron microscopy, 16 powdery mildew species were identified on the basis of morphometrics of fungal structures such as conidial patterns, germination, apresoria, presence or absence of fibrosin bodies, and conidiophores basal cell. Some species identification was confirmed by using DNA analysis of the rDNA ITS region sequences. Most commonly found powdery mildew species were Erysiphe pisi, Golovinomyces cichoracerum, Podosphaera fusca, and P. xanthii. In ornamentals and herbs, Tecomaria capensis was infected with Erysiphe peruviana, rose with Podospahera pannosa, Angelonia augustiolia with Erysiphe quericola and dill with Erysiphe heraclei. Anato, Bixa orellana, was infected by Oidium bixae. In vegetables, for the first time, Podosphaera fusca was identified causing powdery mildew in okra, Abelmoschus esculentus. In addition, two different species of powdery mildew were identified in papaya: Oidium sp. and the common Oidium caricae. In conclusion, Erysiphe heraclei, E. peruviana, E. quericola, Podosphaera fusca, and P. xanthii are new reports for Puerto Rico. In addition, a pilot of an open access online database developed containing information pertaining to powdery mildews of Puerto Rico will allow accurate species identification. This information will help to enforce quarantine regulations to stop the introduction of new species of pathogens in the fragile agricultural ecosystem of the island.

Publications

• Abstracts: Bonilla Aviles, L.L, C. Estevez de Jensen, L. I. Rivera Vargas and B. Roman Aviles. 2011. Identification of powdery mildew in ornamentals and herb plants in Puerto Rico. Phytopathology. 101:S276.
• Latoni-Brailowsky, E. I., L. I. Rivera-Vargas, C. Estevez de Jensen and M. J. Cafaro. 2011. Identification of anamorphic powdery mildews on fruits and vegetables in Puerto Rico. Phytopathology. 101:S277.

Progress 09/01/09 to 08/31/10

Outputs
OUTPUTS: Tropical crops are severely affected by powdery mildews and in certain areas are considered important emerging diseases. Despite of the economic importance of powdery mildews on various crops in Puerto Rico, research focusing in their identification has been insignificant. In the past, several species were identified on the basis of their anamorphic stage by correlating it to their teleomorph and host specificity reported in the literature. A reliable taxonomic determination for most of the powdery mildews is lacking. Emphasizing the importance of gathering knowledge about their identity, beyond the generic Oidium concept, and their biodiversity aspects is fundamental when dealing with exotic plant diseases of quarantine importance. During 2009 to 2010, a survey was conducted at different municipalities around the island of Puerto Rico. Samples were collected to observe the incidence of powdery mildew and identify the causal agent in the different plant hosts. Organic farmers are the most affected by severe powdery mildew outbreaks. Important crops such as mango, tomato, ornamentals, herbs, legumes among other important crops are severely infected at certain seasons of the year. For example, tomatoes were found infected in all areas of the island from November through March whereas some weeds showed infections year round. Powdery mildew species identified on the basis of morphological and/or molecular characterization were: Erysiphe pisi, Erysiphe polygoni, Golovinomyces sp., Oidium neolycopersici, Leveillula taurica and Podosphaera fusca. The most common species is Erysiphe pisi found infecting cilantro, ornamentals, legumes and weed legumes, the former could act as an inoculum source for powdery mildew of leguminous species grown in greenhouses, and serve as alternative hosts for cultivated legumes. Podosphaera fusca was found infecting African marigold. Golovinomyces sp. was found affecting Acalypha wilkesiana, a common ornamental in private gardens in the island. Eryshipe spp. was identified in weeds such as Macroptillium lathyroides and Emilia sanchifolia. In Rosa spp. Podosphaera (Sphaerotheca spp.) was also identified. Leveillula taurica were found infecting peppers and onions. Oidium caricae, O. mangiferae and O. neolycopersici were identified affecting papaya, mango and tomato, respectively. Other fungi associated with powdery mildews identified by sequencing analysis were: Ampelomyces quisqualis, a powdery mildew hyperparasite, Corynespora cassicola, Cladosporium cladosporoides and Leptosphaerulina sp. Failure to extract DNA from several samples will require additional sample collections and evaluation of DNA extraction methodology. PARTICIPANTS: Professional development: Poster presentations were made by two graduate students, Elena Latoni-Brailowsky and Laura Bonilla, at the American Phytopathological Society - Caribbean Division (APS-CD) Annual Meeting joint with the International Integrated Pest Management Meeting held at Nicaragua on project advances. Project leader, Lydia I. Rivera -Vargas, participated as plenary speaker in the XII Mycology Symposium held by the Puerto Rican Society of Mycology at Interamerican University Aguadilla, PR. Project advances were presented. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Combination of DNA analysis with morphological data using scanning electron microscopy will result in a more accurate diagnose of powdery mildew species and their host specificity in Puerto Rico. Establishment of a database of powdery mildew species and their host plants in the island will allow plant pathogen diagnostic clinicians and quarantine inspectors to accurately identify powdery mildews and implement proper management practices in the tropics.

Publications

• Bonilla, L.L., C. Estevez de Jensen and L.I. Rivera Vargas. 2010. Identification of Golovinomyces sp. in Acalypha wilkesiana using Scanning Electron Microscopy and other classification criteria. Phytopathology. (Abstract). Latoni-Brailowsky, E., M. Vaz and L.I. Rivera-Vargas. 2010. Identification of Oidium neolycopersici, a powdery mildew of tomato, using scanning electron microscopy (SEM) and DNA analysis. Phytopathology. (Abstract).