Management, host pathogenicity, and identification of Magnaporthe poae, causal agent of summer patch on annual bluegrass turf.

Graduate student: Melissa M. I. Bassoriello

Principal investigators: Katerina S. Jordan J. Christopher Hall

Project summary:

Summer patch, a root disease caused by the fungus Magnaporthe poae, is most pathogenic on annual bluegrass (Poa annua) golf course greens in North America.  Disease symptom development can occur when the turfgrass is stressed, although M. poae thrives best under conditions of high air and soil temperatures, high soil moisture and high soil pH.

Currently, summer patch disease is managed primarily through preventative chemical applications in conjunction with cultural practices.  However, the appropriate method of fungicide application (e.g. application volume, additional irrigation) and the effects of various cultural practices on pathogen survival and disease development are not well known for the disease on annual bluegrass putting greens.  In addition, pathogenicity and natural resistance in annual bluegrass ecotypes are not known in Ontario.  Finally, proper diagnosis of this disease relies on visual observation of symptoms and signs on the roots, both of which can sometimes be misleading.

The objectives of this study have been to identify M. poae, the cause of summer patch disease, in Canada, develop best management practices for summer patch, and determine host specificity and pathogenicity to gain a better understanding of disease development.

Sept 2012 - UPDATE

Molecular work for identification of Magnaporthe poae in Canada

Magnaporthe poae has been definitively identified in Canada.  A disease note was submitted to the journal Plant Disease and was recently accepted for publication.

Over one hundred (cup-cutter sized) greens samples exhibiting symptoms reminiscent of summer patch were collected from numerous southwestern Ontario golf courses from summer 2009 and 2010.  Several roots of collected samples were covered with dark, ectotrophic runner hyphae and other structures characteristic of M. poae.  Root sections were surface sterilized, plated on antibiotic medium and incubated at 28oC for approximately 2 weeks.  A fungus with morphological characteristics similar to those of M. poae was consistently isolated and used to identify M. poae through molecular techniques (polymerase chain reaction or PCR) and Koch’s postulates. These cultures have been maintained on fresh media through periodic re-culturing.  DNA was extracted from the fungal mycelium of the collected isolates.  To ensure PCR was successful, a small quantity of each PCR product was run on an agarose gel.  As shown in Figure 1, seven DNA bands (~ 520-530 base pairs (bp) in size) were evident and appropriate for M. poae according to previously published literature and personal communication with Dr. H. M. Fouly.

Figure 1. Three DNA bands (~ 520–530 bp) were produced through PCR. (Photo courtesy M. Bassoriello and A. Schwan)

The purified PCR products were sequenced and a BLAST search exhibited seven isolates with 99% and 100% similarity to M. poae in the GenBank database.  As well, pathogenicity of four isolates was confirmed using Koch’s postulates. Healthy P. annua samples (core-sized) collected from an Ontario golf course were inoculated with M. poae-infested grass seed and were placed in a growth chamber at 25-30oC.  After approximately 3 weeks, inoculated plants appeared symptomatic (chlorotic foliage; dark runner hyphae on roots).  Infected roots were sterilized and placed on antibiotic medium; fungal cultures exhibited the same morphology and characteristics as those previously identified as M. poae.

Field trials

A field study was conducted during the 2010 season at the Guelph Turfgrass Institute (GTI) on the Poa annua green established for this project.  The randomized trial consisted of several treatments including a variety of fungicide application regimes and untreated controls.  Four replicates each of preventative application of azoxystrobin (watered-in and not watered-in) and curative application of azoxystrobin (watered-in and not watered-in) were performed.  Various control treatments (no inoculum + no azoxystrobin controls and inoculum + no azoxystrobin) were used. 

Photographs (Figure 2), disease severity ratings, turf quality ratings and visual observation of the roots using core samples were taken during the trial to assess treatments.  As well, cup cutter samples were taken from each plot at the end of the trial. Data analysis using the disease severity ratings (Table 1) and turf quality ratings was completed.  It appears that preventative watered-in application of azoxystrobin is the most effective for pathogen and disease management.

Figure 2. Results of 2010 field trial.

a) no fungicide applied,

b) azoxystrobin application, not watered in

c) azoxystrobin application, watered in. (Photos courtesy M. Bassoriello)

The azoxystrobin trial was re-established in summer 2012, along with newly developed trials examining various nitrogen-based fertilizers (ammonium sulfate, ammonium nitrate and urea) as well as a trial with a combination of cultural management practices (no management, foliar and soil manganese sulfate, azoxystrobin, propiconazole, thiophanate-methyl watered-in, water acidification, azoxystrobin + water acidification, azoxystrobin + managanese sulfate, managanese sulfate + water acidification).  Data collection and statistical analysis is currently underway for the 2012 trials.

M. poae mating and determination of pathogenicity:

The seven isolates that were positively identified as M. poae were mated with each other and with the M. poae ATCC cultures to determine incompatibility and compatibility between fungi.

Figure 3. Photograph of the a) compatibility

and b) incompatibility of various M. poae cultures.

Forty-eight containers (Poa from GTI green) were established and maintained in greenhouse for 3 weeks (allowed roots to grow; maintained at greens height).  Prepared inoculum with ATCC cultures and positive M. poae cultures.  Arranged in randomized complete block design, 4 replicates (Figure 4).  Applied inoculum at soil level and on foliage.  After inoculation, containers transferred to growth chamber with 12-h day/night cycles at 30/25oC.  Containers were trimmed to greens height (~ 3 mm) once every week.  Disease severity was assessed 4 weeks after inoculation using a scale of 0 to 10 corresponding to the percentage of foliar tissue exhibiting chlorosis/dieback:

• 10 --> 100% chlorosis
• 5 --> 50% chlorosis
• 0-->  0% chlorosis

After evaluation, containers were monitored and evaluated once per week, containers were photographed once per week.  Data analysis is currently ongoing.  The growth chamber experiment will be repeated again in 2012/2013.

Figure 4. Preliminary pathogenicity trial.

Fall 2012

The plans for the fall of 2012 are to conduct statistical analyses of data collected from 2012 field trials, collect fresh Poa annua ecotypes and inoculate P. annua ecotypes for further pathogenicity testing, conduct M. poae mating (on wheat; for production of perithecia).

References:

Clarke, B.B., Thompson, D., Murphy, J., and Majumdar, P.  1995.  Development of an integrated summer patch control program for fine turf areas. Proceedings of the Fourth Annual Rutgers Turfgrass Symposium.  pp. 18-19.

Smiley R.W., Dernoeden, P.H., and Clarke, B.B.  2005.  Compendium of turfgrass diseases, 3rd Ed.

Vincelli, P. and Williams, D.W.  2011.  Chemical control of turfgrass diseases.  University of Kentucky Cooperative Extension Service.