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2009. Plant Management Network. This article is in the public domain.
Accepted for publication 30 September 2009. Published 15 December 2009.


A User-Friendly Method to Isolate and Single Spore the Fungi Magnaporthe oryzae and Magnaporthe grisea Obtained from Diseased Field Samples


Yulin Jia, USDA-ARS, Dale Bumpers National Rice Research Center, Stuttgart, AR 72160


Corresponding author: Yulin Jia.  yulin.jia@ars.usda.gov


Jia, Y. 2009. A user-friendly method to isolate and single spore the fungi Magnaporthe oryzae and Magnaporthe grisea obtained from diseased field samples. Online. Plant Health Progress doi:10.1094/PHP-2009-1215-01-BR.


The filamentous ascomycete fungus Magnaporthe oryzae B.C. Couch [formerly M. grisea (T.T.Hebert) M.E. Barr] infects a wide range of monocotyledonous hosts and is the casual agent for a number of economically damaging diseases worldwide including blast of rice (Oryza sativa L.) (1), whereas the morphologically indistinguishable Magnaporthe grisea infects only Digitaria spp. (2). Magnaporthe oryzae infects the rice plant by penetrating directly through the plasma membrane using strong biological turgor pressure and subsequently living on live cells for further invasive growth. The disease lesions begin to appear approximately 3 days after infection, and fill the cells with mycelia. As a result, a typical eye-shaped grey center is visible 5 to 6 days after inoculation in a susceptible rice cultivar, and consequently, panicle blast can be seen post-anthesis (1) (Fig. 1). Although M. oryzae has been studied under extensively for several decades worldwide, descriptions of methods for efficient single-spore isolation from diseased leaves from the field are not available. Techniques to isolate the spores of M. oryzae from different hosts have been passed verbally from one worker to another. The ability to isolate the fungus from field samples is essential for specialists around the globe to study M. oryzae. The objective of this study was to develop a user-friendly method to isolate and evaluate M. oryzae from field samples.


 

 

Fig. 1. Symptoms of rice blast: (A) panicle blast caused by Magnaporthe oryzae and (B) typical eye-shaped lesion. Rice cultivar Banks, from a farm in Clay Co., AR, were used for the pictures.

 

Fungal Isolation

Diseased tissue samples were collected from the rice cultivar ‘Francis’ from an experimental field near Almyra, AR, and from smooth crabgrass (Digitaria ischaemum) growing on the levee of a farm in Stuttgart, AR, USA. Diseased crabgrass leaves or nodes of rice panicles were placed on wet filter papers in a tissue culture container (24 × 24 cm², Fisher Scientific Inc., Pittsburgh, PA) to encourage sporulation. Sterilized pencils were used to support the diseased tissues so that they were in contact with water on the wet filter paper to keep them from dehydrating, but were not submerged, and to reduce the growth of opportunitistic necrotrophic fungi (Fig. 2). The container was covered with a lid and placed under a white florescence light at 21 to 24°C. After incubation for 24 h the tissues were examined under a light microscope to check for sporation of M. oryzae (Fig. 3). Fungal spores were then collected using a small piece of water agar (1 to 5 mm in diameter) on the tip of a microscopic loop (Fisher Scientific Inc., Pittsburgh, PA) (Fig. 3). The captured fungal spores were then transferred to water agar (30% w/v) and incubated for 3 days at 21 to 24°C. Growing hyphal tips were transferred to oatmeal agar (72.5 grams/liter, Difco, Detroit, MI) or alfalfa-rice bran medium for some weakly growing isolates (3), and incubated until spores were produced (Fig 4a).


 

Fig. 2. Induction of sporation of Magnaporthe species (A) from diseased leaf tissues of smooth crabgrass and diseased nodes of rice panicles (B) that had been placed in a container with moistened filter paper and supported with pencils.

 

 


   
  Fig. 3. Transferring blast spores from diseased leaf tissue. Spores (A) were transferred via a small piece of agar attached to the tip of a microscopic loop with the aid of a digital camera connected to a Leica light microscope and visualized on a computer screen (B). Enlargement of the agar attached to the microscopic loop (C).  

 

Fig. 4. Cultures of Magnaporthe oryzae. Colonies after two-day incubation (A); Mycelium on a Whatman filter paper after ten-day incubation (B).

 

Single-Spore Isolation

After sporulation a sterilized loop was used to collect a slice of oatmeal agar or rice bran medium (1 mm in diameter) and spores were suspended in 1.5 mL sterilized water for 1 to 3 min. Ten to 100 µL of the spore suspensions were spread onto oatmeal agaror rice bran medium and incubated for 2 to 3 days. Each mycelial colony from a single spore was collected and prepared for long-term storage. The purified mycelium was grown on Whatman filter paper (70 mm diameter, Schleicher Schuell Bioscience Inc., Sanford, ME) on either oatmeal or rice bran medium for an additional two weeks (Fig. 4b). The filter paper with mycelia and spores was removed from oatmeal or rice bran medium, and placed in a fresh Petri dish, desiccated, and finally stored at -20°C in a small sterilized vial.


Conclusions and Recommendations

In the present study, we presented a detailed method to isolate M. oryzae from field samples. This improved method should aid the effort to study this fungus. Traditionally, single-spore isolations were performed using a specialized microscope making the process time consuming and difficult for each isolation. Successful isolation rate is usually low for new users because the spores are often contaminated with other necrotrophic fungi in diseased tissues. Instead of picking up a single spore under microscope, in the present study, a group of spores were first collected to avoid the issues that commonly arise when attempting to collect a single spore using a microscope. These spores were subsequently separated by plating them onto water agar plates using a method that is commonly used for the isolation of single bacterial colonies.

Magnaporthe oryzae is known as a weak competitor in diseased tissues within fields. Often the diseased tissues are occupied by numerous necrotrophic fungi, hence overnight incubation of diseased tissues to induce sporulation is important to ensure successful isolation. Magnaporthe oryzae is a slow growing fungus on artificial medium. As such, two to three days of incubation on water agar are needed to allow the fungus to develop sufficient mycelium from a single spore. This helps ensure that the sample is not lost. In our experience, the viable mycelia inside living cells were eliminated during surface sterilization. As a result, isolation using surface sterilization has not been effective for M. oryzae. In the present study, the successful rate of pathogen isolation and purification was improved by a two-step process. This procedure was predicted to be applicable to isolates of M. oryzae from any diseased tissues and has been successfully adapted in other laboratories. It was anticipated that specialists around the globe can easily adapt and use this procedure. This spore isolation method could aid efforts to further understand the biology of M. oryzae and to conduct research and develop methods aimed at controlling numerous agronomically important crop diseases worldwide.


Literature Cited

1. Asuyam, H. 1963. Morphology, taxonomy, host range and life cycle of Pyricularia oryzae. Pages 9-22 in: The Rice Blast Disease. S. H. Ou, ed. The John Hopkins Press, Baltimore, MD.

2. Couch, B. C., and Kohn, L. M. 2002. A multilocus gene geneology concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea. Mycologia 94:683-693.

3. Singh, M. P., Lee, F. N., Counce, P. A., and Gibbons, J. H. 2004. Mediation of partial resistance to rice blast through anaerobic induction of ethylene. Phytopathology 94:819-825.