PROJECT RESULTS

Relationship Between Weather Variables and Fusarium Spore Release from Infected Crop Residues

Dr. Jeannie Gilbert

Cereal Research Centre

Agriculture and Agri-Food Canada

Winnipeg, MB  R3T 2M9  Canada

Table of Contents:

• Background and Objective
• Procedure and Project Activities
• Results and Discussion
• Conclusion

ARDI Project:

#01-530

Project Status:

Completed December, 2004

Background and Objective:

Gibberella zeae (Schwein.) Petch, the causal agent of fusarium head blight (FHB), produces perithecia and ascospores, which are the primary source of inoculum.  The conditions under which perithecia develop, and ascospores mature and are released to the environment has been well-documented (Fernando et al. 2000, Trail et al. 2002).  However, little is known about the survival of these ascospores once they have been discharged from the perithecium.  The objective of this study was to examine the release of ascospores in relation to environmental variables.  In addition, an understanding of the effect of the environment on germination of ascospores was undertaken to help refine a model of disease prediction for FHB in Manitoba.

Procedure and Project Activities:

Environmental data for 2003 were recorded every half hour with a Campbell CRI0X datalogger.  Temperature and relative humidity were monitored with a sensor (model HMP35A) installed under a RM Young radiation shield (model 41004-Hmp).  Wind speed and direction was monitored with a Campbell scientific wind set (model 034A-L).  Rainfall was measured with a Texas Electronics rain tipping bucket (model TE525M).  The datalogger was set up May 16, 2003.

Burkard 7-Day Spore Sampler:

In 2003, a Burkard 7-day spore sampler was set up in the centre of an inoculated area.  Spore sampling began on July 14^th and continued until August 11^th, 2003.  The volume of air sampled was 10 litres of air per minute with a tape speed of 2 mm/h.  The tape was changed at weekly intervals, cut into 48 mm sections, and mounted onto glass microscope slides.  Ascospores and macroconidia were identified using a compound microscope (400X) based on size and septation.  The concentration of spores trapped during one hour was calculated by multiplying by 20 the number of spores enumerated on a 100 micron-wide transverse to represent the total number trapped for a one hour period, and by 1.0/0.6 to correct for the estimated number per cubic meter.

Burkard Cyclone Spore Sampler:

A Cyclone Burkard spore sampler was also used to sample airborne ascospores.  Daily collections were made from July 16 to August 12, 2002.  Spores were washed from the cyclone trapping chamber and aliquots plated on to Komada’s medium (selective for Fusarium) for identification of Fusarium species.  The remainder of the wash was spun down to form a pellet.  The water was drained from the pellet which was then frozen at -800°C for DNA extraction and molecular typing for Fusarium species.

Longevity of Discharged Ascospores:

A method to establish the effect of environmental drying was developed to determine longevity of ascospores after discharge from the perithecium, the fruiting body of the fungus that develops in the spring. Gibberella zeae was grown on carrot agar plates under white light in a 20°C incubator for 5 days.  The cultures were then “knocked-down” and placed under UV/white light with a 16/8hr photoperiod for 6-8 days to induce perithecial development.  Once perithecia formed, a new misted Petri dish lid was placed on the culture and left for 2 days under UV/white light.  Ascospores were collected from suspended sterile distilled water droplets on the Petri dish lids.  Initially, ascospores were placed on glass microscope slides and exposed to RH of 30, 60, or 90% at 20°C for 2 or 4 hours and then washed on to water agar plates. After 24 h, germinated and non-germinated spores were counted in random groups of 100 and the average percent germination recorded.  There were 3 fungal isolates, 6 replicates, and the experiment was repeated once.

It was noticed that occasionally the droplet of spore suspension remained intact under 60 or 90% RH for the duration of the experiment.  In such instances it was concluded that those spores were exposed to 100% RH as they were enveloped in water.  In subsequent experiments, 25 µl of spore suspension, collected from Petri dish lids, was placed on the frosted portion of a glass microscope slide, and dried rapidly in the laminar flow-hood to simulate the drying effect that ascospores encounter in nature when discharged.  These spores were then washed on to water agar plates and incubated overnight in the dark.  The average percent germination was recorded. 

Results and Discussion:

During the 4 week sampling period there were 4 periods of rain with accumulations above 5 mm.  Daily maximum and minimum temperatures ranged from 23°C to 34°C and 9°C to 19°C respectively.  Minimum relative humidity (RH) ranged from 18 to 69% and maximum RH from 77 to 99%.  Highest maximum RH was associated with periods of rain, but minimum RH fluctuated following, and even during, rain periods (Figure 1).

Burkard 7-day Spore Sampler:

There was a single major ascospore release during the sampling period.  This occurred after the second rainfall on 18-19^th July (Figure 2). While rainfall patterns indicated that a second ascospore release might have occurred following the 4^th rainfall on August 4-5^th, few ascospores were trapped.  Ascospore release appears to be inhibited by intermittent rainfall and continuous RH of more than 80% (Inch, 2001).  These conditions may have occurred between the first and second rainfall events on the 14^th and 18^th of July respectively, and between the third and fourth events on the 30^th July to 1^st August, and the 4-5^th August, respectively.  The high temperatures after the 4^th rainfall may have prevented ascospore release.  The hourly ascospore release followed a diurnal pattern with low levels of ascospores trapped during the day and increasing numbers being trapped during the evening as RH increased and temperatures dropped (Figure 3).

Burkard Cyclone Spore Sampler:

The Komada’s plates from July 21- 26 and August 4-8 were lost due to a malfunction of the culture fridge.  The results for the remainder of the sampling period are presented in Figure 4. The main Fusarium species trapped by the cyclone spore sampler were F. graminearum, F. equiseti, F. sporotrichioides, F. avenaceum, and F. proliferatum.  Fusarium graminearum was the predominant species trapped, with F. sporotrichioides found at the beginning and end of the sampling period.  Our attempts to use a quantitive PCR assay to measure the ratio of spores of 4 common Fusarium species unfortunately failed.  It appears the amount of DNA of each species in spore form is insufficient to be amplified and to provide an adequate signal with the probes available.  This protocol had been developed for species invading wheat spikes in which the quantity of fungus from mycelium would be much greater (Nicholson et al. 1996).

Longevity of Discharged Ascospores:

Preliminary results are presented as we develop protocols to manipulate ascospores and investigate the effect of relative humidity (RH) on germination.  Results indicated that RH less than 90% and rapid drying led to reduced germination of ascospores.  According to published reports, ascospore discharge starts at approximately 4:00 to 5:00 pm, when the RH is lowest, and peaks around 9:00 pm when RH is 65 to 85% (Fernando et al. 2000).  In nature, ascospores that are successfully deposited on host tissue would have 8 hours of high RH in which to germinate and start to penetrate the substrate.  Future experiments are planned to investigate how long ascospores survive the diurnal drying and rehydration encountered in nature.

Conclusion:

Ascospore discharge follows within 2-3 days of a rain event that is greater than 5 mm, but other factors such as continuous high RH and high temperatures may influence spore release.  Rapid drying of ascospores reduced germination.  We do not know how long ascospores remain viable after diurnal drying and rehydration under natural conditions.

Acknowledgement:

This project was made possible due to funding from the Governments of Manitoba and Canada through the Canada-Manitoba Agri-Food Research and Development Initiative (ARDI).

References:

Fernando, W.G.D., Miller, J.D., Seaman, W.L., Seifert, K., and Paulitz, T.C. 2000. Daily and seasonal dynamics of airborne spores of Fusarium graminearum and other Fusarium species sampled over wheat fields. Can. J Bot. 78: 497-505.

Inch, S. A. 2001. An epidemiological study of fusarium head blight (FHB) of wheat caused by Gibberella zeae. Thesis submitted for degree of Master of Science. University of Manitoba, Winnipeg, Manitoba, Canada

Nicholson, P., Doohan, F., Joyce, D., Rezanoor, H.N., Simpson, D., Smith, P.H., Turner, A, and Weston, G. 1996. Detection and quantification of individual fungal species in Fusarium ear blight by PCR. P 40-46 In Dubin, H.J., Gilchrist, L., Reeves, J. and McNab, A. (Editors) Fusarium head scab: Global status and future prospects. Proceedings of a workshop held at CIMMYT, El Batan, Mexico 13-17 October 1996

Trail, F., Xu, H., Loranger, R., and Gadoury, D. 2002. Physiological and environmental aspects of ascospore discharge in Gibberella zeae ( anamorph Fusarium graminearum ). Mycologia 94: 181-189.

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