Isolation of Fungi from Healthy and Blue-Stain Infected Piñon at the Sevilleta LTER

Zachary T. Gossage1, Joanna Redfern2, Paulette Ford3, William Pockman2, Nate McDowell4, Donald Natvig2, Andrea Porras-Alfaro1, 2, Department of Biological Sciences

1. Western Illinois University, 2. University of New Mexico, 3. Forest Service, 4. Los Alamos National Lab

Piñon trees in piñon-juniper woodlands in the Southwest have been showing increase mortality due in part to major droughts that have been impacting this area. However, it is unclear if drought-induced morality is the sole cause. Under stress conditions, piñon trees are commonly attack by bark beetles (Ips confusus) and blue-stain fungi (Ophiostoma sp.). We described wood-associated fungal communities isolated from cores of healthy and bark-beetle damage trees. Samples were taken from healthy and dying piñon from experimental plots in a piñon-juniper woodland at the LTER site in the Sevilleta National Wildlife Refuge, NM. The experiment utilizes different treatments: excluding 50% of ambient precipitation, 150% mean annual precipitation, exclusion control and environmental controls. Cores were taken from trees, surface sterilize, cut into 1-2 cm sections and plated on PDA (potato dextrose agar) with antibiotics. Isolates were sequenced using the ITS rDNA barcode region. An approximate total of 155 unique sequences were obtained. The wood endophytic communities were dominated by Pleosporales, Dothidiales and Xylariales. Common genera include Penicillium, Hormonema and Pestalotiopsis. Fungi within the genus Ophiostoma were also recovered from bark-bettle damage trees. This initial characterization of wood-isolated fungi will help document potential dormant pathogens and changes in community with the arrival of blue-stain fungi due to insect attack.

>AM427_2 3.1 (sequenced obtained from an insect)CGCGCCTCTCCCCCCCAGGTCCCTTCGGGGCGCCCGCCAGCGGCCGCGAGCCGCCCGAACCTCTTATAAACCGTAACCGAACCGTCTGAGAAACAAACAAAAACAGCCAAAACTTTCAACAACGGATCTCTTGGCTCTGGCATCGATGAAGAACGCAGCGAAATGCGATACGTAATGCGAATTGCAGAATTCAGCGAGTCATCGAATCTTTGAACGCACATTGCGCCCGCCAGCATTCTGGCGGGCATGCCTGTCCGAGCGTCATTTCCCCCCTCAGCGCGCCCTTCTGGGAGCGCTGGCGTTGGGGCTCCTCCGCCCTCTGTGGCGGCAGGGCCCTCAAATCCAGTGGCGGGCCCGTCTGGCTGGCTCCGAGCGCAGTACCGAACGCAAGTTCTCCTCTCGCTTCGTAGCCCCGGCCGGCGCCCAGCCGTCAAGCCGCGCAGGCGACTCTTCCAGGGCCGCCTCGCACTTTTTTACAAGGTTGACCTCGGATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA

Methods:

-Analysis of different types of fungi associated with different sources.

GenBank Hit Closest Genus Order Phylum Other habitats E-value Bit Score Identities %

AF013228 Sydowia Dothideales Ascomycota Various plant hosts 0 1129 99

DQ078308 Pestalotiopsis Xylariales Ascomycota Soil 0 974 99

AY373901 Penicillium Eurotiales Ascomycota Soil 0 1074 99

GQ121322 Alternaria Pleosporales AscomycotaSoil

Carpet, Textiles 0 1109 99

GU566303 Alternaria Pleosporales AscomycotaSoil,

Carpet, Textiles 0 1114 99

FN868479 Truncatella Xylariales Ascomycota Soil 0 1088 99

FN868480 Pestalotiopsis Xylariales Ascomycota Soil 0 854 99

GU244511 Discostroma Xylariales Ascomycota Plant hosts 0 928 98

Most Abundant Endophytes

AcknowledgmentsThis project is currently funded by Forest Service and an WIU URC Summer Grant , DOE and NSF Sevilleta LTER grant.

FUNGAL MOLECULAR ECOLOGY LAB

Blue stain fungi- the plant pathogen: Ophiostoma sp.

Abstract

Dead TreesLiving Trees

Plated on Agar

Cultures Isolated

Sequenced

Experimental Plots:a) Precipitation Exclusionb) Added Precipitationc) Exclusion Controld) Environmental Control

BLAST

Examine

Sampling from:

Core

s

Leav

es

Root

s

Beet

les

Sydowia polyspora Pestalotiopsis sp.

Penicillium canescens Alternaria alternata

Sydowia polyspora

Pestalotiopsis

citrina

Penicillium canescens

Alternaria sp.

Alternaria sp.

Truncatella angustata

Pestalotiopsis

besseyi

Discostroma fuscellum

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InsectCoreRootNeedleTwig

Perc

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ge o

f iso

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Sydowia polyspora was the most common fungal endophyte isolated from twigs, needles and insects. Roots were mainly colonized by a fungus related to Penicillium. Multiple species of Alternaria and Pestalotiopsis were also found in needles and twigs.

Twig

sDistribution of the Most Abundant Fungal Endophytes Distribution of All Isolates Based on Order

Dothideales

Xylariales

Eurotiales

Pleosporales

Hypocreales

Chaetothyriales

Diaporthales

Magnaporthales

Sordariales

Pezizales

Other0

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BarkInsectCoreRootNeedleTwig

Perc

enta

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Ascomycota was the dominant phylum found in P. edulis. At the order level, Dothidiales, Pleosporales and Xylariales are the dominant taxa colonizing twigs, and needles. Roots were mainly colonized by fungi in the Eurotiales and Hypocreales. These orders include a number of fungal pathogens and known endophytic fungi.

Multiple species of Ophiostoma have been shown to be plant pathogens and dubbed ‘blue-stain fungi’ due to the staining they cause on their host’s tissue.

In a 2009 survey of the Piñon-Juniper woodlands in New Mexico, O. montium and O. minus were isolated from dead or dying trees multiple times.

Beshers et al. 2005 measured mortality rates of trees in soil depleted of water after 15 months of drought conditions. Extensive mortality was shown, exceeding 90 percent of Pinus edulis. A relationship among drought, bark beetles (a known pathogen), and fungi was recognized with later research.

S. polyspora, or Hormonema dematoides, is a known endophytic fungi. It has shown potential in producing medically important chemicals such as hormonemate (causes apoptosis) (Filip 2003). Also can be a pathogen causing tip dieback.

P. canescens has been shown to produce xylanase, which could be useful for industrial purposes (Bakri 2003).

A. alternata is a known plant pathogen; causes blight in potatoes and leaf spot disease. This species has been shown to have seven pathotypes that produce host-specific toxins (R Hatta et. al 2002).

Pestalotiopsis contains fungi that are very difficult to distinguish at the species level. They generally don’t show host-specificity. Most species have been identified as plant pathogens and the rest are saprobes or endophytic fungi (Wei 2004).

(http://www.mycology.adelaide.edu.au/images/Alternariq.gif)

(Allen 2007)

(http://www.bcrc.firdi.org.tw/fungi/fungal_detail.jsp?id=FU200802260010)

(Filip 2003)

(http://people.ucsc.edu/~ggilbert/images_lab/pestalotiopsis.jpg)

Preliminary analysis of fungal sequences using BLAST Three mortality in the southwest

Ophiostoma sp. fruiting bodies

Piñon infected with Ophiostoma