CHARACTERIZATION OF POPULATIONS OF ENDOPHYTIC FUNGI IN IMPORTED PLANT PRODUCTS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1009659
• Project Start Date: Jul 1, 2016
• Project End Date: Apr 4, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UTAH STATE UNIVERSITY
(N/A)
LOGAN,UT 84322

Performing Department
Biology

Non Technical Summary
Much of the fresh produce that is consumed in the United States is imported from other countries. Because of the potential to introduce plant pathogens with imported plant products, government agencies such as USDA-APHIS are charged with protecting American agriculture by enforcing quarantine laws and by screening produce before it enters into the country. In most cases, the sheer volume of produce that must be screened implies that only material with clearly visible disease symptoms will be intercepted. Yet, virtually all land plants harbor endophytic fungi, some of which could be pathogenic species. Thus the potential exists to inadvertently introduce new pathogens with healthy-looking produce. This project uses a combination of molecular and morphological methods to examine the fungi that are entering the country cryptically within imported fruits and vegetables.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	2410	1102	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
2410 - Cross-commodity research--multiple crops;

Field Of Science
1102 - Mycology;

Keywords

mycology

plant pathology

endophyte

dna

fungus

agricultural import

Goals / Objectives
The primary goal of this project is to characterize the populations of endophytic fungi in fruits and vegetables that are imported into the United States. There are three objectives for this project: 1) Characterize the populations of endophytic fungi that are being brought into the United States with selected imported fruits and vegetables. 2) Compare the endophyte populations in selected US-grown fruits and vegetables with those in imported material of the same species. 3) Determine whether populations of endophytic fungi differ between organically and nonorganically grown products for both imported and domestically produced crops. Anticipated deliverables are scientific publications or abstracts that indicate which fungi are present in selected plant products that are imported into the United states or compare these with fungi present in domestically grown produce.

Project Methods
Fresh fruits and vegetables that are readily available to shoppers in markets will be chose for the work done under objectives one and two. Both imported and domestically grown products representing the same species will be studied and the geographic source of the material will be recorded. An emphasis will be placed on things such as melons, grapes, or certain vegetables that are consumed in large volumes and that are both imported and grown in the continental USA. The third objective will be accomplished by comparing endophyte populations in fruits and vegetables grown organically and nonorganically both within and outside of the United States. A culture-based approach will be used to identify endophytes present in these plant materials. Healthy tissue will be removed under laboratory conditions from the samples and cut into multiple small sections. These sections taken from the produce will be surface-sterilized in 95 percent ETOH for 5 seconds, washed in 5 percent sodium hypochlorite for 2 min, and washed in 70 percent ETOH for 2 min before finally being rinsed in sterile water and blotted dry on sterile paper. Each of the surface-sterilized pieces will checked for surface contamination by pressing both sides on a control plate of malt extract agar before being incubated on a second malt extract agar plate. Plates will be incubated at room temperature for up to 6 weeks to allow endophyte growth. All fungi growing from the leaf tissue will be isolated into pure culture. In any case where the control plates produce microbial growth from the surfaces of the samples, the plates and corresponding pieces of tissue will be discarded. Colony morphology will initially be used to group the isolates from field samples into rough morphospecies and any of the endophytic fungi that form fruiting structures in pure culture will be identified using standard morphologically-based methods. When morphological methods do not permit identification, molecular approaches will need to be used to identify many of the cultures. To do this, DNA will be extracted from the mycelia of these fungi and the Internal Transcribed Spacer (ITS) region will be PCR-amplified using well known fungal-selective primer sequences (ITS4 and ITS5). After amplifying the target sequence, it will be purified for sequencing using a commercially available purification kit. After the purification, sequences for the ITS region of the fungi will be generated at the Center for Integrated Biosystems on the USU campus. The fungi will be identified by using their ITS sequences to do BLAST searches against the bank of sequence data at the National Center for Biotechnology lnformation - GenBank. Because many of the ITS sequences in Gen Bank were generated from environmental samples, they do not have Latin names associated with them - instead they are assigned accession numbers and treated as unnamed Operational Taxonomic Units (OTUs). For the work proposed here, the Gen Bank numbers that most closely match the sequence data from the endophytes cultured will be used as identifiers for the fungi.

Progress 07/01/16 to 04/04/19

Outputs
Target Audience: Nothing Reported Changes/Problems:Bradley Kropp retired from USU, December 31, 2018. This project has ended. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Nothing to report.

Publications

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:USDA-APHIS mycologists, plant pathologists Changes/Problems:Except for the early end to the work due to retirement, no changes were made to this project. As was reported last year, a problem encountered during the current reporting period was that the endophytic fungi obtained in culture usually failed to produce identifying fruiting structures. What opportunities for training and professional development has the project provided?No new undergraduate research projects were done with this project during this reporting period. How have the results been disseminated to communities of interest?No new articles were produced during this reporting period. What do you plan to do during the next reporting period to accomplish the goals?None. This work is being discontinued prior to its end-date because the PI has retired.

Impacts
What was accomplished under these goals? The work that had begun on grapes in 2016 was expanded to include some other plant products during this reporting period. One of the comparisons made for this period was of the endophytic fungi found in organically and nonorganically grown avocados imported from Mexico. The results indicated that 43 and 42 percent of the cultures, respectively, yielded fungi. Overall, however, there was very little difference between the organically and nonorganically grown avocados from Mexico. A total of 8 fungal taxa were cultured from the organic avocados, whereas 7 were obtained from the nonorganic material. Some differences were noted in the abundance of certain fungi between the organic and nonorganic avocados, but further work needs to be done to determine whether this is statistically meaningful. A comparison was also made between organically grown green onions that were either imported from Mexico or produced in California. Very few endophytic fungi were cultured from either set of onions. Only three fungal taxa were isolated into culture: two from the organic and two from the noinorganic onions, with one fungus being shared between the organic and nonorganic material. Cucumbers that were either grown nonorganically in the US or organically grown and imported from Mexico were also compared. Similar to what was observed with the onions, very few endophytic fungi were found in cucumbers. Only 2 fungal taxa were cultured from these samples, both from the organically grown Mexican material and no fungi were cultured from nonorganically grown domestic cucumbers.

Publications

Progress 07/01/16 to 09/30/16

Outputs
Target Audience:Target Audience USDA-APHIS and state agency inspectors, plant pathologists Changes/Problems:Changes/Problems No changes have been made to the project. One problem has been encountered in that the endophytic fungi growing from the materials studied thus far have often failed to produce fruiting structures that would permit identifications to be done. What opportunities for training and professional development has the project provided?Opportunities One undergraduate student (Meghan Graham) is currently involved in this project. She has done an undergraduate research project based on the work that was done on grapes described above. She has had an opportunity to gain hands-on experience with sterile culture methods and other basic microbiology techniques. How have the results been disseminated to communities of interest?Dissemination No published articles have yet been produced. A poster will be presented by the undergraduate researcher in the project at an upcoming Biology Department undergraduate research symposium. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work The plan of work for the upcoming year is to focus primarily on objectives one and two of the project. The products to be sampled will depend on what is seasonally available at any given time throughout the year.

Impacts
What was accomplished under these goals? Accomplishments This is a new project that began in July 2016. The emphasis thus far has been directed at objective two of the project: Compare the endophyte populations in selected US-grown fruits and vegetables with those in imported material of the same species. The early work for the project has been mainly focused on comparing populations of endophytic fungi from grapes. Fungi were cultured from samples of grapes that had been imported from Chile and that had been produced in the United States. The fungal species involved still need to be identified. Preliminary results indicate that four different fungi had been imported in grapes from Chile whereas six were present in one population of US-grown grapes and a locally grown sample yielded only two different fungi. There was minor overlap in the endophyte populations within imported and nonimported grapes. Two fungi cultured from chilean grapes were also cultured from US-grown grapes.

Publications