assessing the reaction of american wildrice to inoculated pathogens

Assessing the Reaction of American Wildrice to Inoculated Pathogens.Assessing the Reaction of American Wildrice to Inoculated Pathogens.Raymond Porter*, Robert Nyvall, and Laura CareyRaymond Porter*, Robert Nyvall, and Laura CareyUniversity of Minnesota, NCROC, 1861 E. Hwy 169, Grand Rapids, MN 55744University of Minnesota, NCROC, 1861 E. Hwy 169, Grand Rapids, MN 55744

INTRODUCTION

American wildrice (Zizania palustris— Fig. 1) has several

known stem rot diseases, caused by the fungal

pathogens Nakataea sigmoidea (Fig. 2), Bipolaris oryzae,

and B. sorokiniana. The Bipolaris species also cause the

foliar diseases fungal brown spot and spot blotch (Fig. 3).

Since these diseases have previously been the target of

selection to improve varietal resistance, methods have

been developed to inoculate leaves with conidia

suspensions to develop resistant varieties. Selection for

resistance to stem rots in wildrice has not been done.

In this study, methods were developed for growing

inoculum of N. sigmoidea and for delivering known

quantities of propagules of all three species to the stems

of wildrice varieties. These methods were applied in a

multilocation trial, where plots of a single variety were

inoculated with all three species to test several fungicides

for efficacy in reducing the effects of these diseases. This

experiment was used as an opportunity to develop

methods to quantify differences in disease severity for

future assessment of varietal resistance in a breeding

program. Specific objectives included:

1) Develop and assess the effectiveness of a method of

production and inoculation of N. sigmoidea, and

2) Assess the effectiveness of leaf and stem disease

rating methods in detecting statistical differences

produced by different treatments (fungicides).

MATERIALS AND METHODS

Variety: Itasca (a high-yielding, shattering resistant

variety selected for resistance to fungal brown spot.

Design: RCBD, 1.5 x 3.0 m plots, 8 fungicide treatments,

6 reps at each of 3 locations.

Inoculation and treatments: Eighteen isolates of B.

sorokiniana were cultured on a medium consisting of

course perlite, wildrice flour, and 1% PDA (1:2:4).

Separately, 18 isolates of B. oryzae were cultured on

same medium. Conidia were filtered into suspension

with water after 15 (B. sorokiniana) or 18 (B. oryzae)

days of growth. Inoculum of B. sorokiniana was applied

at a rate of 4.5 million conidia per plot with a CO2

sprayer at mid-tillering. B. oryzae was applied 5 days

later at 1.8 million conidia per plot.

Six N. sigmoidea isolates were cultured on the same

medium. Mature sclerotia were produced in 14 days, at

which time medium was dried, crushed, and filtered

through a #10 seive (Fig. 4) to produce the dried

inoculum (750 sclerotia/ml). At late tillering, inoculum

was spread with a Spred-Rite granular applicator on the

water surface just inside the side borders of each plot,

at 16,000 sclerotia per plot (Fig. 5).

Five fungicides—Tilt (propaconizole), Headline

(pyraclostrobin), Quadris (azoxystrobin), Quilt

(azoxystrobin+propaconizole), and Stratego

(trifloxystrobin+propaconizole) were applied at boot

stage, and two of these were also applied at heading,

making up seven fungicide-timing treatments plus a

control.

Disease assessment and analysis:

All plots at a location were harvested on the same

day. Ten stems and 20 flag leaves per plot were

collected at harvest and frozen to be later rated for

area affected by disease. Diseased leaf area was

estimated by comparison with the Clive James key

for Septoria leaf blotch (Key 1.6.1). Diseased stem

area was estimated without the aid of a key. Two

pieces were cultured from each stem and one piece

from each leaf (a total of 20 each of stem and leaf

pieces). Five to seven days later, fungal species were

identified for each lesion to estimate the incidence of

each pathogen. Indexes were calculated for leaf and

stem diseased area due to a particular pathogen by

multiplying the visual estimate by the frequency of

the pathogen incidence on cultured leaf pieces.

Each location was analyzed separately using SAS

Proc Mixed, adjusting for spatial variability with the

function sp(powa). Significance of pairwise

comparisons at P<0.05 was used as the criterion for

declaring treatments statistically different.

Fig. 1 Cultivated American wildrice, Zizania palustris cv. 'Petrowske Purple'

Fig. 2 Sclerotia inside of a wildrice stem infected with Nakataea sigmoidea

Fig. 3 Fungal brown spot (left), caused by B. oryzae, and Spot blotch caused by B. sorokiniana.

Fig. 4 Granular inoculum of N. sigmoidea in perlite medium to be filtered (left). Sclerotia amid perlite granules in inoculum (right).

Fig. 5 Granular applicator used to apply dry inoculum of N. sigmoidea (left). Granules on water surface adhering to wildrice stem (circled in photo on right).

Fig. 6 Estimated diseased leaf area of flag leaves for treatments at Aitkin. Treatments having a letter in common are not significantly different at P=0.05.

RESULTS

Significant treatment differences were found for

diseased leaf area at all three locations, but the Aitkin

location was especially severe, with many significant

comparisons (Fig. 6). Incidence of B. oryzae was far

greater than B. sorokiniana in leaf lesions and also

showed significant differences (not shown). When

multiplied by diseased leaf area, the index magnified

treatment differences (Fig. 7).

Fig. 7 Index estimating percentage of leaf area diseased due to B. oryzae at Aitkin.

photo by Dave Hansen, MAES

Stem lesion area was difficult to estimate, and

analysis showed no significant treatment differences.

B. oryzae was predominant among the stem lesions,

and N. sigmoidea did not have as high an incidence

as expected (1-3% vs 10-30% for B. oryzae). However,

when stem lesion area was multiplied by incidence of

all three stem pathogens combined, significant

treatment differences were seen (Fig. 8).

Fig. 8 Index estimating percentage of stem area diseased due to Bipolaris spp. and N. sigmoidea at Aitkin.

CONCLUSIONS

1. Because incidence of Nakatae in stems was low,

this method of inoculation needs to be improved.

Earlier application of inoculum and development

of a method to apply sclerotia directly to stems are

priorities for future research.

2. Indexing of both diseased leaf and stem area with

pathogen incidence in cultured lesions improves

statistical discrimination between treatments. This

may indicate that a significant number of lesions

(both stem and leaf) are of non-fungal origin.

ACKNOWLEDGMENTS

Funded through USDA-ARS Cooperative Agreement No. 58-

3640-4-122. Wildrice growers Tom Godward, Rod Skoe, and Ed

Mohs graciously provided space for experiments. Dan Braaten

and Henry Schumer provided technical assistance.

Representatives of BASF, Bayer, and Syngenta provided

fungicides for the treatments.

photos by James Percich, U of MN

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