Diagnosis and control of winter squash storage rots in western Oregon

Final report for GW18-157

Project Type: Graduate Student
Funds awarded in 2018: $25,000.00
Projected End Date: 03/31/2019
Grant Recipient: Oregon State University
Region: Western
State: Oregon
Graduate Student:
Major Professor:
Dr. Kenneth Johnson
Oregon State University
Expand All

Project Information

Summary:

Winter squash could be an important locally-grown winter food and provide critical winter
income to farmers in western Oregon if it could be successfully stored. In the Willamette Valley,
severe storage losses currently prevent sales through winter markets. For example, in reputedly
long-storing cultivars, losses from 50-100% have been observed by December. Little is known
about the causal agents of these uncommonly severe rots and their prevention. Consequently,
this project will characterize causes of squash fruit rots and evaluate practical methods to reduce
their incidence. A preliminary investigation of squash rots has shown that Fusarium culmorum is
frequently associated with diseased fruits. Interestingly, this fungus is known as a floral pathogen
of grasses and other monocots. Oregon’s Willamette Valley is a major grass seed production
area, and grasses are common cover crops for diversified farmers. Consequently, the hypotheses
are that F. culmorum is abundant due to regional cropping patterns, it is successfully rotting
squash because this fungus infects flowers, and the common floral-end rots of squash are caused
by pathogens that colonize flowers. In field inoculation trials, I will confirm the pathogenicity of
F. culmorum and other fungi associated with squash rots. I will conduct field experiments at
research and grower farms to evaluate if fruit rots are reduced by a) dryland production, b) nongrass
mulches (via mechanisms of reduced pathogen inoculum and unfavorable environmental
conditions for fungal dispersal and colonization), and c) chemical control. I will evaluate squash
storability over time in a closed farm bay, which is the most profitable storage method for the
region. This project will provide a profile of an important disease and methods to reduce its
incidence, which will increase profitability of winter squash through a direct reduction of
postharvest fruit losses.

Project Objectives:

Objective 1. Identify the most significant pathogens reducing winter squash storability in
the Willamette Valley
To develop management plans for a disease problem, a clear diagnosis of the pathogens
involved is necessary. To do this, we will employ molecular and morphological identification
techniques as well as in-field pathogenicity trials.
Hypothesis: We hypothesize that F. culmorum as well as other organisms previously identified,
(other Fusarium spp., Botrytis spp., Phoma spp. and Sclerotinia sclerotiorum) are rotting squash
fruit, but that F. culmorum causes the early-in-storage and most devastating losses.
Protocol: Identification of potential winter squash rot pathogens Winter squash from the 2017
growing season with representative rots have been cultured for fungi from diseased areas.
Systematic symptom descriptions characterizing the date at which the rot started, as well as the
color, location (floral-end, stem-end, side of fruit) and severity (percent squash area rotted) of the
rot have been recorded. Morphology and DNA sequencing of the Internal Transcribed Spacer
and Elongation Factor 1 alpha gene regions are being used to identify the fungi. Following rot
characterization, we will better understand which of the pathogens rot fruit early/middle/late in
the storage season, and where the pathogen infects the fruit. Floral-end rots are likely initiated
from flower infection (in the field during the growing season), while rots beginning at the stemend
and the side of the fruit are more likely due to pathogens present on the surface of the fruit
with the rot initiated via wounds created during handling (harvest).
Determine if F. culmorum and/or other important squash rotting fungi colonize flowers.
In summer 2018, we will plant 3 plots of susceptible winter squash varieties ‘Sunshine’ and
‘Bonbon’ in an irrigated field with a vetch mulch to control for rain splash of naturally occurring
soilborne pathogens onto flowers. Female, fruit-bearing, squash flowers will be sprayed with
liquid suspensions of potential pathogens identified from 2017. Potential pathogen inoculation
treatments will include a broth-only control, F. culmorum, and other consistently-associated fruit
rot pathogens from characterizations described above. Flowers will be sprayed after opening and
monitored throughout the season for deterioration, fruit abortion and rot symptoms after fruit set.
Fruit from inoculated flowers will be taken to the lab and cultured to confirm infection occurred
from the inoculated fungi. This will complete the diagnosis of the fruit rot pathogens and articles
will be prepared for dissemination to growers and scientific journals, following conclusion of the
experiment.
Objective 2. Evaluate the efficacy of dryland production, organic mulches and chemical
control for the suppression of fruit storage rots.
In-season management strategies that are easily implemented and cost effective are
important to winter squash profitability. To evaluate different management strategies, we will
plant on-farm trials in the growing season of 2018.
Hypothesis: We hypothesize that squash grown in dryland plots will have less disease and store
longer than squash grown in irrigated plots. We also hypothesize that a vetch mulch will reduce
disease incidence in irrigated plots because the mulch acts as a physical barrier, suppressing
splash of soilborne squash rotting fungi onto the flowers. We hypothesize that a cereal mulch
will increase rot disease incidence in irrigated plots because F. culmorum residing in the cereal
mulch will splash onto flowers. We hypothesize that a chemical spray at bloom will reduce the
ability of the pathogen to infect flowers and reduce disease incidence.
Protocol: Dryland versus Irrigated Production effects on storage rot incidence To compare
storability of squash grown under dryland and irrigated production, we will plant field trials at
the Oregon State University Vegetable Research Farm, Gathering Together Farm, and 47th St.
Farm. At all locations, there will be dryland and irrigated plots of our two representative,
susceptible squash cultivars ‘Sunshine’ and ‘Bonbon.’ At all locations and in both irrigated and
dryland experiments, there will be three replicates of 5 plants of each variety planted in 10-footlong
plots in rows 60 inches apart. Winter squash
will be direct seeded into dryland plots and
transplanted into irrigated plots. Plants will be
grown through the season and fruits harvested for
storage at 45 days after pollination. Individual fruit
weights and fruit number will be recorded.
Mulching effects on storage rot incidence
To compare methods for disease reduction,
additional plots in the irrigated and dryland fields
of ‘Sunshine’ and ‘Bonbon’ will be mulched using
cereal mulch and vetch mulch. Plots will be
mulched at planting. As above, there will be three
replicates of 5 plants of each variety planted in 10-
foot-long plots in rows 60 inches apart.
Transplants or seed will be sown into the mulches depending on irrigation production of the plot.
Plants will be grown through the season and fruits will be harvested for disease evaluation and
storage at approximately 45 days after pollination. Individual fruit weights and counts of
harvestable fruit will be recorded. Figure 4 shows an example of a field plot.
Chemical control effects on storage rot incidence To determine the efficacy of fungicide
sprays as a control of storage rots, a set of plots at the Vegetable Research Farm will be planted.
Figure 4. Field experiment map. Gray is
control, yellow is cereal mulch, green is vetch
mulch. Plots such as this will be repeated at all
three farms in irrigated and dryland fields.
As above, there will be plots in both the dryland and irrigated fields. And as above, there will be
three replicates of 5 plants of each variety planted in 10-foot-long plots in rows 60 inches apart.
These plots will be sprayed with prothioconazole (Proline 480 SC) at the rate of 5.7 fl. oz./acre
using backpack sprayers. Plots will be sprayed twice, once at first bloom and again 14 days later.
Plants will be grown through the season and fruits will be harvested for disease evaluation and
storage at approximately 45 days after pollination. Individual fruit weights and counts of
harvestable fruit will be recorded.
Storage Trials Fruit grown at the Vegetable Research Farm will be placed in bins and
stored in a closed barn bay maintained at >32F and ambient humidity [5,10]. In OSU storage
trials this has been shown to be the most profitable storage environment [9,10]. Fruit grown at
Gathering Together Farm and 47th Ave. Farm will be placed in bins and stored in a shaded
greenhouse or barn maintained at >32F and ambient humidity. In November, January and March,
evaluations of the number of rotted fruits and the color and location of rot will be recorded.
Disease symptoms will be photo-documented (see Scholarly Publications section). Diseased
fruits will be removed from storage as they are detected.
Statistical analysis Storage data will be analyzed using the statistical program R.
Regression models comparing yield to irrigation practice, mulch type, squash variety and rot
incidence and severity will be analyzed.
Profit analysis Following storage trial results, final yields and total potential profits,
using average market prices from GTF and 47th Ave. Farm will be determined. Cost of labor and
management techniques will be compared and an extrapolated cost of management and
profitability bulletin will be developed for dissemination at outreach events.
Educational Outreach Plan
Objective 3. Engage growers in project planning, activities and outcomes
Many farmers have inspired and informed this project. Both project farmers (GTF and
47th Ave Farm) have partnered with OSU personnel in the past on squash and other projects. We
will present results of our trials at field days and grower meetings.

Cooperators

Click linked name(s) to expand
  • Dr. Alexandra Stone (Educator and Researcher)
  • Dr. Amy Garrett (Educator and Researcher)

Research

Materials and methods:

Objective 1. Identify the most significant pathogens reducing winter squash storability in the Willamette Valley

Identification of potential winter squash rot pathogens.  In fall 2017, fungi were cultured  from rotted areas of squash fruit. Symptom were characterized by date rot started, color, location (floral-end, stem-end, side of fruit) and severity. Fungal morphology and DNA sequencing of barcoding regions, Internal Transcribed Spacer (ITS) and Elongation Factor 1a, were used to identify fungi. Four species of Fusarium: F. culmorum, F. incarnatum-equiseti, F. oxysporum, and F. solani, identified from fungal collections were grown on Spezieller Nahrstoffarmer agar (SNA) plates to produce inoculum. Healthy C. maxima squash were surface sterilized with 15% bleach and 11 fruits per isolate were inoculated. A sterilized one cm diameter cork borer was used to make a 0.5-cm deep wound ineachwinter squash. Plugs of each fungal isolate on SNA were cut with a 0.8-cm cork borer and placed in the wound, mycelium side down, and sealed with petroleum jelly. Control fruits (11 total) were mock-inoculated with a plug of sterile SNA. After 2 wk., rot appearance, diameter and depth were recorded. Fungi were re-isolated and identified to confirm Koch’s postulates.

Determine if F. culmorum and/or other important squash rotting fungi colonize flowers.In summer 2018, forty 2-plant plots (4 ft. plot) of rot-susceptible winter squash cv. ‘Sunshine’ were established in an irrigated field with fabric mulch to control for irrigation splash of naturally occurring rot pathogens. Female, fruit-bearing, squash flowers were sprayed with liquid suspensions (105 spores/liter) of F. culmorum (2 isolates), F. solani, F. incarnatum-equiseti, and a broth-only control. Flowers were sprayed the day they opened and monitored throughout the season. At maturity, fruits were harvested, counted and weighed. Fruit from inoculated flowers was stored in a closed barn bay at >32F ambient humidity. As rot symptoms developed, fungi were re-isolated to confirm identify of inoculated pathogen.

 

Objective 2. Evaluate the efficacy of dryland production, organic mulches and chemical control for the suppression of fruit storage rots.

Mulching effects on storage rot incidence. An additional twelve 5-plant (10 ft.) plots in the irrigated field of ‘Sunshine’ were mulched at planting with straw or fabric (6 of each). A set of six plots were sprayed with prothioconazole twice at flowering according to label specifications. Six control plots did not receive any mulch or chemical application. Plants were grown through the season and fruits were harvested and stored as described above.  In November, December, and January, evaluations of the number of rotted fruits and the color and location of rot were recorded. Diseased fruits were removed from storage as they were detected.

 

Research results and discussion:

Identification of potential winter squash rot pathogens. 96 fungal isolates were cultured from 19 rotted C.maximawinter squashin 2017. On average, 12% of fruit surface area was rotted (severity ranged from 1% to 60% rotted surface area). Fusarium species were the most common fungi isolated from rotted fruit accounting for 72% of cultures(Table 1). They also were commonly associated with calyx-end rot symptoms. Fusarium culmorum and F. solani produced the most severe symptomsin inoculation assays. F. incarnatum-equiseti also produced consistent lesions. F. oxysporum was the least aggressive among inoculated fungi. Mock-inoculated controls did not show any rot symptoms(Fig. 1).

Determine if potential pathogens colonize flowers. For each of the five inoculation treatments, 55 flowers were inoculated (275 total). 59 fruits in total were harvested. In storage, rot symptoms began to appear on October 11th, and continued through March 1st. The treatments with the most rotted fruit were F. solani (63%), F. culmorum 1and the control(50% of harvested fruit; Table 2). Very few fruits exhibited blossom-end, pink to white rot, though re-cultured fungi match the morphology of those inoculated. This may be due to the liquid inoculation method – the suspension may have impacted other environmental factors that help contribute to inoculation.

Evaluate efficacy of dryland production to suppress storage rots. The field planting of dryland squash was unsuccessful due to extreme spring weather relative to the planting date of the experiment. Therefore, the efficacy of dryland production to reduce storage rot was unable to be evaluated. Late dryland plantings of winter squash on fabric mulch are too dry and hot for the establishment of squash plants.

Evaluate efficacy of organic mulches to suppress storage rots.The mulched field plots of ‘Sunshine’ squash yielded between 4 and 8 fruits per plot. Straw mulch plots had lower yields than fabric, chemical treated or control plots, likely due to seeds in the straw growing and becoming weeds and therefore competing with squash plants for nutritional resources (mean yield 3.15 kg per plot, P<0.05; Table 3). Fruits in storage began to rot on October 11thand continued through March 1st. Control plots had the highest average percentage of rotted fruit per plot (59.4%) while fabric mulched plots had the lowest average percentage of rotted fruit (37.1%; Table 3). Four fruits showed the characteristic blossom-end rot and all of them were from control plots, suggesting that mulching does reduce the incidence of inoculum splash onto flowers. This should be taken into consideration in locations where winter squash storage rot has been a problem. Straw mulch can reduce storage rot disease incidence, but farmers should be aware that sprouting from the mulch can reduce overall yield. Chemical control, if available to a conventional grower, provides high yields and can reduce disease incidence to levels similar to fabric mulch compared to control plants (Table 3).

Table 1. 2017 survey of fungi associated with rotting C. maxima winter squash fruits in storage between October and December.

Fungusy Percent Isolatedz Rot color associated with fungus
Fusarium culmorum 11.46 Pink-White Rot
Fusarium incarnatum-equiseti 22.92 White Rot
Fusarium oxysporum 13.54 Pink-White Rot
Fusarium solani 6.25 White-Gray Rot
Fusarium sp. 17.71 White Rot
Penicillium sp. 7.29 Green Rot
Other Fungi 20.83 Black, Yellow, or White Rot

yFungi cultured from rotting squash tissue, isolated in pure culture and identified morphologically and molecularly using ITS and EF1 gene regions. zPercentage of each species out of all identified fungi. Figure 1.Mean surface area (cm2) rotted of mock-inoculated C. maxima squash fruits at 2 weeks after inoculation with a fungal isolate. Colors depict different Fusariumspecies along with an agar-only control. Isolates were collected from surveys of rotted winter squash in 2017 growing season.

Table 2. Counts of harvested fruits and rotting fruit from the field-grown, fungus-inoculated flowers of C. maxima cv. ‘Sunshine’ plants in 2018.

Inoculation Fungus Fruit Harvested Fruit Rottedz Percent Rottedz
Control 12 6 50
F. culmorum 1 8 4 50
F. culmorum 2 12 5 42
F. solani 16 10 63
F. incarnatum-equiseti 11 2 18

zCount and percentage of fruit rotted on March 1st, 2019.

 

Table 3.Mean counts of harvested fruit and yield per plot in the 2018 irrigated mulch field trial of C. maxima cv. ‘Sunshine’ plants.

Treatment Fruit Harvested Fruit Rotted Percent Rotted Yield (kg)
Straw 4.2 + 0.48z 2.2 + 0.48 50.8 + 7.57 3.2 + 0.65
Fabric 5.7 + 0.49 2.0 + 0.37 37.1 + 7.62 6.0 + 0.65
Chemical 6.5 + 0.43 2.7 + 0.76 39.3 + 10.15 7.0 + 0.52
Control 5.7 + 0.33 3.3 + 0.33 59.4 + 6.27 5.5 + 0.43

zMean + standard error.

Participation Summary

Educational & Outreach Activities

1 Journal articles
5 Webinars / talks / presentations

Participation Summary:

70 Farmers
70 Ag professionals participated
Education/outreach description:

This work resulted in a first report of Fusarium culmorum causing fruit rot of winter squash in Oregon in the journal Plant Disease. Two posters at scientific meetings and three grower talks were presented to scientists, agriculture professionals, and farmers. Talks were 30 – 50 minutes long. An extension publication for PNWhandbooks.org describing the disease is in progress.

Project Outcomes

1 Farmers intend/plan to change their practice(s)
1 Grant received that built upon this project
Did this project contribute to a larger project?:
Yes
Project outcomes:

This project helped us better understand the inoculation mechanism of certain organisms that cause a significant storage rot in winter squash. This will contribute to future sustainability by providing a starting point for research efforts into other management strategies or pathogen biology. Our work on mulch as a management strategy will reduce storage rot losses and weeding labor costs. Overall, this project helps contribute positively to future sustainability in winter squash production.

Knowledge Gained:

My knowledge about sustainable agriculture changed during the course of this project. I learned about the timing of a dryland farming practice and how difficult it can be if it is improperly delayed. I also learned the about mulching with straw to control inoculum. It became weedy and would require additional labor to maintain. I think that my awareness towards sustainable agriculture projects was increased and I plan to look into additional sustainable agriculture solutions to challenges in vegetable production.

Information Products

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.