Paper towel roll method modified for the detection of Sclerotinia sclerotiorum in bean seeds
SUMMARY
The white mold of the bean, caused by the fungus Sclerotinia sclerotiorum (Lib.) Of Bary, is one of the main diseases of the culture. The pathogen can be disseminated by infected seeds, which play an important role in infestation of new planting areas and establishment of disease at the beginning of the crop cycle. This paper presents an adaptation of the paper roll method, originally developed for the detection of Colletotrichum lindemuthianum, with the objective of detecting the presence of S. sclerotiorum in bean seeds. In this method, the seeds were incubated for 7 days at 20 ° C in rolls of paper towel for germination and were kept under conditions of 100% relative humidity. After this period, infected seedlings and dead seeds, surrounded by mycelium characteristic of S. sclerotiorum, were transported to gerboxes on two sheets of moistened filter paper. After 3 to 4 days of incubation at 20 ºC and under a regime of 12 hours of light for 12 hours of dark, the sclerotia were observed in the seeds and seedlings. The method was relatively fast, simple and inexpensive, as well as having the advantage of simultaneous detection of S. sclerotiorum and other important pathogens transmitted by bean seeds, such as C. lindemuthianum, Macrophomina phaseolina and Rhizoctonia solani.
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ABSTRACT
White mold, caused by the fungus Sclerotinia sclerotiorum (Lib.) Is one of the most damaging diseases of beans. Dissemination of the pathogen by infected seeds is important because it can infest new planting areas and the disease may establish in the beginning of the crop cycle. The present work describes an adaptation of the seed health test using the germination paper towel method, originally developed for the detection of Colletotrichum lindemuthianum, for assaying the presence of S. sclerotiorum in bean seeds. The test consisted of placing bean seeds in germination paper towels, which were rolled and placed in the germination chamber and kept at 100% relative humidity and 20 ° C for seven days. After this period, the infected seedlings and the dead seeds surrounded by a white cottony growth were collected and placed in a gerbox over two wet filter papers. The sclerotia characteristic of the pathogen appeared around the seeds after four days of incubation. This method is relatively rapid, inexpensive, and has the additional advantage of detecting simultaneously other important pathogens in bean seeds, such as Colletotrichum lindemuthianum, Macrophomina phaseolina and Rhizoctonia solani.
The white mold of the bean, caused by the fungus Sclerotinia sclerotiorum (Lib.) Of Bary, is one of the main diseases of the crop, causing severe damages in the winter plantations in irrigated areas (2, 6). The dissemination of the fungus can occur by ascospores, which are released from the ascas and ejected in the air, by the sclerotia transported in the irrigation water and / or associated with the seeds, and by means of infected seeds, which play an important role in the infestation of indigent areas and in establishing the disease at the beginning of the culture cycle (2, 11). Infestation of new areas is a serious problem, since sclerotia can remain viable for up to 8 years in the soil (11). Due to the important role of seeds in the spread of the disease, Machado (5) and Menten (7) proposed the zero tolerance pattern for S. sclerotiorum in bean and soybean seeds of the basic, certified and supervised classes.
The fungus S. sclerotiorum occurs at relatively low incidences of bean and soybean seeds, rarely exceeding 2% (10), therefore the methods for their detection must be sensitive, reproducible and, in seed production systems, it is also essential that they be cheap and fast . The filter paper method, with seed incubation for 30 days at temperatures between 5 and 7ºC under continuous dark, is recommended by the Rules for Seed Analysis (3) to verify the presence of the fungus in bean seeds. However, several other methods also showed sensitivity for the detection of S. sclerotiorum . Menezes (6) recommends, in addition to observation of the lot to verify the presence of sclerotia, the incubation of the seeds by the filter paper method, for a period of 10 to 15 days, time necessary for the formation of sclerotia. Koch & Menten (4) found that the filter paper method with incubation for 14 days at 15 ° C under continuous dark was also suitable for the detection of the pathogen. Peres (10) developed the semi-selective medium (NEON), on which the seeds are incubated for 7 days at temperatures between 14 and 20ºC in the dark, the pathogen being detected by the change in color of the medium, which occurs in the presence of acidic substances. S. sclerotiorum is found in seeds because it produces oxalic acid .
The paper towel roll method, originally developed by Anselme & Champion (1), is recommended for the detection of Colletotrichum lindemuthianum (1, 3, 6) , a pathogen that causes easily observable spots on cotyledons, but can also detect those that affect germination, such as Macrophomina phaseolina and Rhizoctonia solani (6). This method, with a modification, has been used since 1993 in the Central Laboratory of Seeds and Seedlings (LCSM) of the Coordination of Integral Technical Assistance, with the main objective of detecting C. lindemuthianum in bean seeds, but also has evidenced the presence of S. sclerotiorum and the other pathogens mentioned above . This modification allowed the detection of S. sclerotiorum in 17 and 18 samples in 1993 and 1994, when 437 and 618 samples were analyzed, respectively (8, 9). All lots in which the pathogen was detected came from production fields in which the white mold had occurred, indicating a relation with the field history. In the other fields the disease was not observed and also the fungus was not observed in the seeds.
The method of the modified towel paper roll consisted of the distribution of seeds on two sheets of paper towel for germination, previously moistened, measuring 38 x 28 cm. Fifty seeds were distributed on two sheets of paper towel being covered by another of the same paper. The leaves were folded in the direction of the largest dimension and rolled perpendicular to the first. The paper rolls were placed vertically and incubated for 7 days in a 20 ° C chamber in plastic bags, to maintain moisture during incubation. In each sample 400 seeds (5) were analyzed. For the detection of S. sclerotiorum, seedlings with symptoms of infection ( Figure 1 ) and dead seeds, both surrounded by cotoneous mycelium , were removed from the rolls, placed in a gerbox containing two sheets of moistened filter paper, incubated under 12 hours of fluorescent light and 12 hours of dark at 20 o C for 3 to 4 days. The sclerotia characteristic of S. sclerotiorum developed on the roll itself, or more frequently were formed after incubation, confirming the presence of the pathogen in the seeds ( Figure 2 ).
The modified roller method was compared to that described by Koch & Menten (4), in order to measure its sensitivity. A bean seed sample from a moderately infested field was divided into three sub-samples. The first two were distributed in rolls of paper towel and tested according to this new proposed methodology, but the rolls were incubated in different environments, being 8 in wood germinator, which maintains 100% relative humidity, and the other 8 were conditioned in plastic bags and placed in a chamber, being periodically re-emitted. The third sub-sample was incubated in Petri dishes for 14 days in continuous dark (4). The fungus was detected in 2.24 and 2.88% of the seeds kept in the germinator and in the BOD chamber, respectively, and in 1.10% of those incubated in the plates. The statistical analysis did not show significant differences between the results, indicating that the proposed method presents similar sensitivity to the method described by Koch & Menten (4), used for measurement.
Eventually the proposed method may overestimate the incidence of the fungus if it spreads from infected seeds to neighboring ones. This fact, however, is of little importance for S. sclerotiorum, whose tolerance standard is zero. The method has the advantage of making it possible to detect S. sclerotiorum by means of a simple, low sensitivity and cost-sensitive procedure that can be easily applied in routine analyzes and can also be used for the simultaneous detection of other pathogens important for culture of common bean, such as C. lindemuthianum, M. phaseolina and R. solani.