© 2002 Plant Management Network.
Fungi Associated With Postemergence Cotton Seedling Disease in Missouri
J. A. Wrather and B. Phipps, Professors in the Plant Science Unit, University of Missouri, P. O. Box 160, Portageville, MO 63873; and C. S. Rothrock, Professor of Plant Pathology, University of Arkansas, Plant Science 217, Fayetteville, AR 72701
Wrather, J. A., Phipps, B., and Rothrock, C. S. 2002. Fungi associated with postemergence cotton seedling disease in Missouri. Online. Plant Health Progress doi:10.1094/PHP-2002-0722-01-RS.
Surveys were conducted to identify fungi associated with postemergence cotton seedling disease in Missouri. Samples consisted of 10 cotton seedlings, 2 to 3 weeks after emergence, with symptoms of seedling diseases collected from a 0.25 ha area in each of 60 fields in 1997 and 1998. Four genera of fungi were cultured from the roots (Fusarium, Pythium, Rhizoctonia, and Thielaviopsis) and three species were identified: Rhizoctonia solani, Thielaviopsis basicola, and Pythium ultimum. Rhizoctonia solani, T. basicola, and P. ultimum were cultured from seedlings in 70%, 47%, and 15% of fields sampled in 1997, respectively, and 55%, 17%, and 5% of fields sampled in 1998, respectively. Repeated tests of pathogenicity confirmed that R. solani AG-4, T. basicola, and P. ultimum were major causal agents of postemergence cotton seedling disease in Missouri. This study provides the first documentation on the distribution and frequency of fungi associated with postemergence seedling disease complex of cotton based on a survey of randomly selected fields.
Seedling diseases caused more estimated yield loss during the last 20 years than any other diseases of cotton in Missouri (10). Estimated yield loss due to seedling diseases in Missouri over this period ranged from a low of 590 × 103 kg of lint in 1989 to a high of 3266 × 103 kg of lint in 1997.
Farmers in Missouri employ one or more practices to reduce the incidence and severity of cotton seedling diseases. They may plant on raised beds, plant high-quality seed that has been treated with fungicides, plant only when the five-day forecast is for warm and dry conditions, and/or apply fungicides in the seed furrow at planting.
Several fungicides are labeled by the Environmental Protection Agency for application to the seed furrow, i.e., infurrow, at planting for management of various cotton seedling diseases. The labels for most of these fungicides indicate that they are for control of cotton seedling diseases caused by one genus of pathogenic fungus. Combinations of fungicides are often used by farmers to control seedling diseases of cotton caused by a broad spectrum of fungi. Infurrow fungicide use based on the pathogen present in a field may be more efficient than routine use of fungicide combinations, and cost savings may be realized by targeting treatments to diseases known to be present.
Fungi that most commonly are associated with cotton seedling diseases include species of Pythium, Fusarium, Rhizoctonia, and Thielaviopsis (4). Current knowledge of the agents causing these diseases has been based on sampling intentionally selected fields. No comprehensive survey on the presence and geographic distribution of the causal agents of postemergence seedling disease of cotton has been conducted. The objective of this study was to determine the distribution and frequency of fungi associated with postemergence seedling disease of cotton in Missouri based on a survey of randomly selected fields.
Surveys were conducted in 1997 and 1998 to identify the fungi colonizing cotton seedling roots and hypocotyls in Missouri. Maps of the six counties where cotton is grown in Missouri were each divided into 30 equal-sized areas (180 areas total), and seedlings were collected from one cotton field near the center of 60 randomly selected areas (9). Samples were only collected from fields where infurrow fungicides had not been used. Two seedlings were collected from each of 10 arbitrarily selected locations within a 0.25 ha area of each field 2 to 3 weeks after emergence, and 10 arbitrarily selected seedlings with symptoms of seedling disease were kept for analysis. These seedlings were stored in a plastic bag at 2 to 4°C and transported to a laboratory for processing that day. Roots were excised from seedlings at ground level, rinsed for 5 min in running tap water, surface disinfested by immersion in 0.5% NaClO for 1.5 min, rinsed in sterile water, and blotted dry with paper towels. Symptomatic tissue was plated on water agar (2%) and incubated at 22°C. Growth of all colonies on water agar at this temperature was slow. After 96 h, hyphal tips of all fungal colonies growing from roots were transferred separately to potato-dextrose agar (PDA), and the roots were transferred to a selective medium for isolation of T. basicola (8). The identification of Pythium, Rhizoctonia, and Thielaviopsis species was based on colony and/or spore morphology. Nuclear status of all Rhizoctonia isolates was determined by staining hyphae with DAPI (4', 6'-diamidino-2-phenylindole). All isolates of R. solani cultured from seedlings in 1997 were separated into 26 morphologically distinct groups. Each of these was characterized for anastomosis group (2).
Pathogenicity Tests of Cultures
Five representative isolates of each R. solani anastomosis group identified and five representative isolates of P. ultimum cultured from seedlings were selected for pathogenicity tests. Cotton plants, cv. Stoneville 474, were grown in previously autoclaved field soil in 12-cm pots at 22°C in a 12-h photo period. Fifteen seeds were planted in each pot, and the seedling populations were thinned to 10 per pot 7 days postemergence. Ten days after emergence, a 4-mm-diameter plug of PDA and mycelium of R. solani or P. ultimum was placed next to the hypocotyl of 5 seedlings in each pot, 0.5 cm below the soil surface. A plug of PDA without the pathogen was placed next to the hypocotyls of the other 5 seedlings. Plants were harvested 10 days later, and the hypocotyls and roots were observed for symptoms of disease. Each isolate of R. solani and P. ultimum and the control was replicated 4 times. The experiment was repeated.
Five representative isolates of T. basicola cultured from seedlings were randomly selected for pathogenicity tests. Two-week-old cultures of T. basicola on 6 PDA plates were blended for 2 min at low speed, mixed with 1000 g autoclaved soil, and distributed into 1-liter cardboard cartons. Two-week-old sterile plates of PDA were blended, mixed with autoclaved soil, and distributed into 1-liter cardboard cartons to serve as controls. Fifteen seeds of cv. Stoneville 474 were planted into the soil in each container and incubated at 22°C in a 12-h photoperiod. Plants were harvested 10 days after emergence, and the roots and hypocotyls were observed for disease symptoms. Each carton was a replicate, and each isolate test for pathogenicity was replicated 4 times. The experiment was repeated.
Pathogens Cultured from Seedlings
Cotton seedlings colonized by P. ultimum were rare in Missouri during 1997 and 1998 (Table 1), but the isolates collected from these seedlings were pathogenic. The reason P. ultimum was rarely found is unclear. Pythium ultimum may have been causing preemergence seed rot and seedling death, but data on incidence of preemergence seed rot and seedling death were not collected. Pythium ultimum was only cultured from seedlings from fields in the northern most cotton growing area of Missouri. The daily soil temperature 10 cm deep in this area averaged 4°C less than the soil temperature in the southern most cotton growing areas of Missouri during the study period. These cool soil temperatures probably enhanced the incidence of seedling disease due to P. ultimum. This pathogen should be considered a threat to cotton seedlings in the northern most cotton growing areas of Missouri. We did not collect data on incidence of preemergence seed rot and seedling death, and we are not sure of the threat it poses. As a precaution, farmers should continue to plant seed treated with a fungicide to manage this disease.
Table 1. Plant-parasitic fungi cultured from roots of cotton seedlings with symptoms of seedling disease in Missouri during 1997 and 1998.
a Ten seedlings with symptoms of seedling diseases were collected from a 0.25 ha area in each of 60 fields in Missouri in both of 1997 and 1998 and were analyzed for pathogenic fungi colonizing the damaged tissue. Six hundred seedlings were analyzed in each of 1997 and 1998.
Seedlings colonized by T. basicola were found each year (Table 1). This pathogen was found in 47% of fields surveyed in 1997 and 17% of fields surveyed in 1998. All of the representative isolates of T. basicola tested were pathogenic. This is the first report of black root rot on cotton in Missouri and the northern most occurrence of the disease. This pathogen should be considered a threat to cotton seedlings in Missouri.
Rhizoctonia solani was cultured from cotton seedlings collected from 70% of fields surveyed in 1997 and 55% of fields surveyed in 1998. Of the 26 morphologically distinct groups of R. solani cultured in 1997, six groups did not anastomose with any of the tester isolates, two groups were identified as AG 2-1, 13 groups were identified as AG-4, 4 groups were identified as AG-7, and one group was identified as AG-11. AG 2-1 and AG-4 are commonly associated with seedling diseases on numerous crops (6). AG-7 is considered a group with limited pathogenicity. It has been found in southeast Asia (3) and has been recovered from soybean and rice in Arkansas (7) and cotton in Georgia (1). AG-11 has only recently been characterized. It was recovered from lupine in Australia and has been recovered from soybean and rice in Arkansas (3). This is the first time AGs of R. solani from Missouri have been characterized. Rhizoctonia solani AG-11 did not cause symptoms on cotton roots or hypocotyls in growth chamber tests, but all isolates of AG-4, AG-7, and AG 2-1 tested caused reddish brown lesions on roots and hypocotyls.
Fusarium spp. were cultured from more than 50% of the cotton seedlings collected each year (Table 1). Seedlings colonized by Fusarium spp. were found in samples from all fields surveyed in both years. Further research is needed to identify the species of Fusarium cultured from cotton seedlings in Missouri and the pathogenicity of these species.
Rhizoctonia solani, T. basicola, and P. ultimum were cultured occasionally from seedlings from the same field. During both years, a single plant-parasitic fungal genus was cultured from seedlings collected from 63% of fields, and multiple parasitic fungi were cultured from seedlings from 37% of fields surveyed. We are not sure why these three pathogens were not detected in seedlings from every field. Johnson et al. (5) determined that the presence of some cotton root pathogens in soil was related to crop rotation. Crop rotation may have influenced the pathogens present in some fields, but we did not collect crop rotation history.
Weather conditions and several biotic and abiotic factors in soil may influence the incidence and severity of cotton seedling diseases. The results of this survey were similar each year it was conducted, but weather conditions may cause differences to occur in subsequent years.
Several fungi have been reported as causal agents of cotton seedling disease in the United States, and these reports were based on seedlings collected from intentionally-selected fields. Results of surveys based on samples from intentionally selected fields or samples submitted to a clinic are inherently biased. This study provides the first documentation on the distribution and frequency of fungi associated with postemergence cotton seedling diseases based on a survey of randomly selected fields.
Rhizoctonia solani and T. basicola were the fungal species most often associated with cotton seedling diseases in Missouri. Fusarium spp. were frequently cultured from cotton seedlings throughout Missouri, but further research is needed to identify the species of Fusarium cultured from seedlings and the pathogenicity of these cultures. The most useful methods for management of postemergence cotton seedling diseases in Missouri should be those targeted at these fungi.
This research was, in part, supported by the Missouri Agriculture Experiment Station.
We thank Mrs. Joyce Elrod for her efforts in this project. This project was supported by the National Cotton Foundation.
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