Impact of fungal root endophytes on Verticillium wilt using cucurbits as model hosts

CSANR Project 161

Status: Complete

Project Summary

V. dahliae (Vd) is a soilborne fungus that infects a broad range of economically important plants. Infection causes chlorosis and necrosis and yield loss due to invasion and colonization of vascular tissues. The pathogen’s wide host range makes crop rotation challenging for disease reduction. Soil fumigation is economically and environmentally costly and must be done repeatedly at high rates due to the resilience and long-term survival of the microsclerotia. Endophytes are fungi and bacteria that live asymptomatically within plant tissue, and can be isolated from almost all plants. Endophytes provide valuable services including protecting plants from root disease-causing organisms. Although the benefits that bacterial endophytes contribute to its hosts are increasingly recognized, less is known about the benefits of fungal endophytic associations. One study observed that melons colonized with Cadophora species were able to reduce infection by Fusarium oxysporum f. sp. melonis and another found that actinomycete species were able to suppress Pythium aphanidermatum infection of cucumber.  In Washington, pumpkins and other cucurbits are high-value specialty crops. Pumpkins are very susceptible to Vd and infection results in yield loss and plant death. This project’s objectives are to (i) identify endophytes in cucurbit roots in WWA, (ii) assess Verticillium wilt (VW) potential on pumpkin and cucurbit grafting rootstocks associated with specific endophytes, and (iii) determine if Vd antagonism is due to the competition, parasitism, or metabolite production by endophytic fungi. This project addresses novel approaches to disease management.

In June 2016, pumpkin cv. Cinnamon Girl, and watermelon cv. Sugar Baby, and squash cv. Tetsukabuto were transplanted into an organically-managed field experiment that was naturally infested with 11 cfu/g soil Verticillium dahliae at WSU Mount Vernon NWREC. The objectives were to: (i) isolate and identify endophytes from cucurbit roots in western Washington, (ii) determine whether isolated endophytes are antagonistic to V. dahliae and antagonism is due to the competition, parasitism, or metabolite production, and (iii) assess Verticillium wilt potential on pumpkin and cucurbit grafting rootstocks when inoculated with endophytes or non-inoculated. Roots (three plants for each of three cultivars in three replications) were excavated, surface disinfested, and plated onto corn meal and modified potato dextrose agars. In total, 603 fungi were
recovered of which 118 isolates were V. dahliae. Of the remaining 485 remaining, the highest number of endophytes (186 of 485, or 38%) were isolated from moderately susceptible ‘Cinnamon Girl’ pumpkin while the lowest number (139 of 485, or 28%) was isolated from susceptible ‘Sugar Baby’ watermelon. Amplification and sequencing of the ITS region of select morphotype isolates revealed Actinomucor sp., Chaetomium sp., Clonostachys sp., Dendryphion sp., Diaporthe sp.,
Epicoccum sp., Fusarium equiseti, Microdochium sp., Schizothecium sp. These nine endophytes were assessed for antagonism against V. dahliae in an assay in which an agar plug of V. dahliae and an endophyte were plated on opposite sides of an mPDA plate and allowed to grow towards each other. Colony expansion of V. dahliae was less when plated against an endophyte, for all fungal endophyte treatments, compared to plates with V. dahliae only. Antagonism to V. dahliae was also assessed in plantae. No significant differences in disease severity, or plant/ root fresh or dry weights
were observed among plants inoculated with Chaetomium sp., Epicoccum sp. Microdochium sp., or Schizothecium sp. regardless of being challenged with V. dahliae or not. V. dahliae is a poor competitor in the soil and a poor root colonizer when certain endophytes are present, potentially increasing plant resistance to infection.

Annual Entries

2016

Principal Investigator: Debra Inglis
Additional Investigator: Lydia Tymon
Progress Report: http://csanr.wsu.edu/wp-content/uploads/2017/10/161Inglisrevised.pdf
Grant Amount: $29,966

Publications

A fact sheet (#FA2106-01) entitled ‘What is an endophyte’ was published online in April 2016 at
https://ag.tennessee.edu/biodegradablemulch/Documents/what_is_an_endophyte_formatted.pdf.
A poster on endophytes and frequency of isolation of endophytes was presented at the 2016 WSUMount
Vernon NWREC Field Day in July. In November, a workshop was organized by Dr. Lydia Tymon
for the Seattle Tilth Conference in Wenatchee, WA. The workshop, entitled ‘Alternatives for Managing
Verticillium dahliae on Vegetables’ was a one hour, three-part lecture with approximately 35
attendees. ‘Fungal Root Endophytes as a Potential for Biological Control’ was presented by Dr. Lydia
Tymon.

Additional Funds Leveraged

USDA NIFA SCRI CAPS Grant-- Grafting watermelon: A new sustainable management practice for
soilborne disease and a new value-added enterprise for Washington ($42,386, $10,000 to Tymon).
Northwest Ag Research Foundation—A potential alternative for managing Verticillium dahliae in
cucurbit cropping systems in western Washington ($5,400).

Impacts and Outcomes

• Short-Term:
While the benefits of bacterial endophytes are increasingly recognized, less is known about the
benefits of fungal endophytes. This project provided an opportunity to learn about potential fungal
endophytes which have not been sufficiently studied in cucurbit production systems, especially in
fields that have never been fumigated. Of the likely fungal endophytes identified to genus in this
study, many have been documented as antagonistic to plant pathogens in previous studies. For
example, Clonostachys sp. has not only been documented as a mycoparasite (Xue 2003) but some
isolates also produce argadin, which is a chitinase inhibitor (Noriko et al. 2000). In another study,
Alternaria longipes, Epicoccum nigrum, Phomopsis sp., and Trichoderma atroviride exhibited
significant inhibition of Rhizoctonia solani (Lahlali and Hijri 2010). Antagonism to R. solani by A.
longipes and E. nigrum was shown to be due to the production of volatile compounds while T.
atroviride was a mycoparasite of R. solani and Phomopsis outcompeted R. solani. It is noteworthy that
isolates of Clonostachys, Alternaria, Epicoccum, and Trichoderma were recovered as endophytes
during this study. The species of these selected isolates are yet to be determined and it is unclear
whether isolates from this study will utilize the same mechanisms of antagonism to V. dahliae as
described in other studies. However, these next steps are anticipated, and needed, for future projects.
Cultivar was not a significant factor nor was root section in the frequency of isolation of the fungi
collected and although not significant, differences in frequency were observed. ‘Sugar Baby’, a V.
dahliae susceptible cultivar, had the lowest frequency of isolation, and the fewest number of fungi
isolated from the root tips whereas ‘Tetsukabuto’ squash, a highly resistant V. dahliae cultivar, had a
higher frequency of isolation and the greatest number of fungi isolated from the root tips. Verticillium
dahliae infection is initiated when microsclerotia germinate in response to root exudates and
penetration into host roots occurs at the root tips (Klosterman et al. 2009). This pathogen is known
to be a poor competitor in the soil (Barron and Fletcher 1970) and has been observed to be a poor
root colonizer when certain endophytes are present (Pantelides et al. 2009). Possibly, the higher
number of endophytic fungi colonizing the root tips as detected in our study may be contributing
factors to the resistance that ‘Tetsukabuto’ exhibits towards V. dahliae. In a previous study, seedlings
inoculated with a non-pathogenic isolate of F. oxysporum had reduced disease severity when
seedlings were planted in V. dahliae infested soils (Gizi et al. 2011).
Four endophyte inoculants appeared to increase Verticillium wilt tolerance in ‘Cinnamon Girl’
pumpkins. Disease severity, plant fresh and dry weights, and root dry weights were not significantly
different for plants whose roots were associated with Chaetomium sp., Epicoccum sp., Microdochium
sp., and Schizothecium sp. prior to planting in either V. dahliae-inoculated or non-inoculated soils,
especially when inoculated with Chaetomium sp. In addition, plants inoculated with both Chaetomium
sp. and V. dahliae had Verticillium wilt disease severity ratings, plant fresh and dry weights, and root
dry weights that were similar to non-V. dahliae, non-endophyte inoculated plants. This endophytic
association provided the added benefit of increased plant growth along with increased disease
resistance and this reaction has been previously described in barley where plants inoculated with
Chaetomium sp. had greater root fresh weight, although not shoot weight, than non-inoculated plants
while also reduced powdery mildew severity (Vilich et al. 1998).

• Intermediate-Term:
Host was not a significant factor in determining frequency of isolation of fungi, which suggests that
endophyte associations with roots may be interspecific. Commercial production of grafted seedlings
for vegetable transplants could benefit if endophytes isolated from one grafting rootstock can
associate with a number of different rootstocks and scions, potentially increasing tolerance or
resistance to V. dahliae. Moreover, endophytes could be used as amendments in potting mixes
when grafting of the scion takes place thereby allowing rootstocks to form the necessary association
with the endophyte prior to transplanting.

• Long term:
Reducing Verticillium wilt in field settings will potentially enhance the vegetable grafting and/or
cucurbit transplant industries by minimizing costs for fumigation, as well as the negative impacts
that fumigation has on the environment. Additionally, crop rotations could be shortened if
endophyte colonization can limit root colonization and V. dahliae reproduction, thereby reducing
primary inoculum in the field. Currently, for V. dahliae management, it is recommended that fields
should be rotated with less high value crops such as pea or cereals for 3 years before planting again.
Reducing crop rotation by even 1 year could represent significant savings to a grower especially in
areas like western Washington where available land for crop rotation is limiting.