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A simple method for detection of Phytophthora nicotianae from soybean soil

A simple method for detection of Phytophthora nicotianae from soybean soil A high detection rate of Phytophthora nicotianae was obtained from the naturally infested soil of soybean fields. Air-dried soils were firstly moistened in a flask and then pre-incubated at 25 °C for 1­4 weeks before flooding with distilled water and baiting with soybean leaf disks for 6, 12, and 24 h. The baits were then thoroughly washed, flooded with 10­15 ml of distilled water in Petri-dishes and incubated under continuous fluorescent light for 72 h. Sporangia started to emerge from the margins of leaf disks. They were easily observed under the stereomicroscope. Pure culture of the fungus was obtained by spreading zoospore suspension on a 1.5% water-agar containing anti-bacterial antibiotics. This is the first report of recovery of P. nicotianae from naturally infested soybean soils using sensitive leaf disks of soybean (Williams cultivar) as bait in Iran. All isolates were determined to be A2 mating type. Key words: Phytophthora; Glycine max; detection; baiting technique; mating type INTRODUCTION Baiting techniques use plant material to isolate selectively the pathogen from soil or diseased roots. The baiting technique is commonly used for detection of Phytophthora occurrence in soils (Erwin and Ribeiro 1996, Mohammadi et al. Abbas Mohammadi, Department of Plant Pathology, University of Birjand, Birjand, Iran Amohammadi@birjand.ac.ir 2008). The technique involves floating pieces of susceptible tissue on a soil water slurry with a high water/soil ratio (Erwin and Ribeiro 1996). Zoospores formed by Phytophthora in the sample infect the baits which can be detected by plating baiting tissue onto selective agar containing antibacterial and antifungal antibiotics allowing outgrowth of Phytophthora from the tissue (Reeser et al. 2011). Phytophthora species growing out of the bait can be identified on the basis of colony morphology, mycelial characteristics, cardinal growth temperatures, and production, morphology, and dimensions of sporangia, oogonia, and antheridia, or DNA sequence analysis (Lamour and Kamoun 2009). Depending 29 Journal of Agrobiology, 29(1): 29­32, 2012 on the time of year at which the sample is taken, the efficiency of detection ranges from 0 to more than 90%. The host species from which the bait tissue is derived also influences the efficiency of detection (O'Brien et al. 2009). Although the bait tissue is derived from a host species that is susceptible to the pathogen, different host species give very different efficiencies of detection (Erwin and Ribeiro 1996). Isolation of Phytophthora from the infected bait requires a considerable time, the use of selective media and considerable knowledge of the genus(Yamak et al. 2002). A major problem is the presence of fast growing organisms such as Pythium, which tend to inhibit growth of the target species (Canaday and Schmitthenner 1982). The infection of the bait can be a limiting factor. The efficiency of detection of Phytophthora in soil by the baiting technique can be improved by drying out the soil and rewetting it (double baiting demonstrating that, as with tissue sections, the pathogen, although present, will not always grow out of the sample (Canaday and Schmitthenner 1982). Davison and Tay (2005) found that double baiting increased the recovery of positive samples. The quality of the water used can also significantly affect the outcome as zoospores are very sensitive to toxic ions present in unpurified water (Tsao 1983). The objectives of this study were to develop a rapid and sensitive baiting method for isolation of P. nicotianae from the soil carried with transported soybeans or in production fields, and to develop a rapid assay to detect the pathogen in infected soybean fields. were floated immediately on the surface of the water. After 6, 12, and 24 h, the floating leaf disks were removed, washed, and placed in another Petri dish with 25 ml of sterile distilled water. Sporangia emerging from the edge of the infected leaf discs were observed under a stereomicroscope after 48 h of incubation in the water. Leaf discs with sporangia of Phytophthora were placed in another Petri dish with 10 ml of sterile distilled water. The entire set of Petri dishes was placed in a refrigerator at 4 °C for 30 min and then placed back at 25 °C for 1 h to complete the process of zoospore release. The zoospore solution was spread on 1.5% water agar containing pentachloronitrobenzene at 25 g ml­1, carbendazim at 25 g ml­1, rifampicin at 20 g ml­1, and ampicillin at 50 g ml­1. The germinated zoospores were isolated 12 h later and pure cultures were obtained (Erwin and Ribeiro 1996, Hua-Bo and Xiao-Ming 2003). Cultures were grown on 5% clarified carrot agar and maintained with regular transfers. Vegetative and reproductive stages were examined in cultures grown for 1 to 4 weeks. Isolates were scored for appearance of hyphae, size, and shape of sporangia and chlamydospores, and the presence or absence of oospores. Observations were compared to published descriptions for Phytophthora species (Ho 1981, Erwin and Ribeiro 1996, Ranjbaran et al. 2006, Gallegly and Hong 2008). The mating types of isolates were determined by pairing known A1 and A2 testers of P. nicotianae on 10% clarified V8 juice agar medium. The unknown isolate was placed on one side of the 60-mm-diameter Petri dish and the known A1 or A2 tester was placed on the other side. The Petri dishes were incubated in the dark at 25 °C for 4 to 8 weeks or until oospores were formed. The mating type was identified for unknown isolates based on the presence of oospores. If pairing with the known A1 tester produced oospores, then the unknown isolate was determined to be A2, and vice versa. If isolates did not produce oospores after 8 weeks, a second attempt was made using previously identified P. nicotianae testers (Ranjbaran et al. 2006). Inoculums for pathogenicity tests were prepared by growing isolates on oatmeal agar in Petri plates at 25 °C for 2 weeks. Inoculations were performed by the standard hypocotyls method (Mohammadi et al. 2008), using 2 × 2 mm pieces of mycelia, and the wound was covered to prevent desiccation of the inoculums and host tissue. Ten 7-day-old seedlings of soybean MATERIALS AND METHODS Soil samples were collected from soybean fields from Golestan, Mazandaran, and Lorestan provinces, Iran, during growing seasons. Five individual samples were taken arbitrarily to a depth of 15 cm using a hand trowel and were well mixed to represent a single composite sample for each field, with a final volume of approximately 100 cm3. All soil samples were air dried, triturated, and then stored in paper pockets at room temperature. P. nicotianae isolation from soil was made by modification of the soybean leaf baiting method (Canaday and Schmitthenner 1982, Hua-Bo and Xiao-Ming 2003). Soil samples were moistened, preincubated at 25 °C for 1­4 weeks, and then flooded with sterile distilled water. Twenty leaf disks from the Williams soybean seedlings Journal of Agrobiology, 29(1): 29­32, 2012 were inoculated with each isolate and grown in the greenhouse at 25±2 °C with supplemental fluorescent and incandescent light (Mohammadi et al. 2008, Wu et al. 2011). sample (Gallegly and Hong 2008). Soybean leaf disk is a specific baiting method to P. sojae (Canaday and Schmitthenner 1982). Despite this host specificity, these baits also attract Pythium species. This study showed that soybean leaf disks baiting were effective for isolating P. nicotianae from soybean soils. Prewetting of soil for various periods of time before submersion in water favours the production of sporangia. Other studies showed that prewetting soil before dilution plating or baiting with pepper leaf disks favoured the detection of oospore inoculums of P. capsici. Double baiting increased the recovery of positive samples from 1.9 to 2.5% and 6.3 to 7.5% of samples taken from the centre and margins respectively of disease fronts in Western Australia (Davison and Tay 2005). When leaf disks are floated for a short time (about 1 h), P. sojae is more frequently detected than other fungi, but at longer incubation times (12 h), P. nicotianae is also detected. At incubation times longer than 24 h, Pythium species are also detected on leaf disks. Ten isolates from Phytophthora species that were detected from infected soybean soils from Iran were P. nicotianae. This is the first record of P. nicotianae on soybean in Iran. This data showed that P. sojae is the major causal agent of soybean stem rot in Iran (Mohammadi et al. 2008). The second species is P. nicotianae. All isolates were detected as being the A2 mating type. Ranjbaran et al. (2006) showed 95% of P. nicotianae from Iran were the A2 mating type. Information on distribution of A1 and A2 mating types is critical to determine the genetic variability within the field pathogen population. Only one-tenth of the Virginia tobacco fields had both mating types, and all those samples originated from fluecured tobacco fields, indicating a low genetic diversity (Parkunan et al. 2010). The relative ratios of A1 and A2 mating types could suggest the level of sexual recombination occurring in natural field conditions, as well as the potential for oospore production. Predominance of a single mating type in the majority of Iran soybean fields may indicate genetically less diverse populations (Ranjbaran et al. 2006). P. nicotianae isolates were sent to another research group in the Tarbiat Modares University in Iran. Their results with specific primers (par A1) were in agreement with our morphological observation and detected them as P. nicotianae. They studied one isolate from California and 25 isolates from twenty hosts by ten RAPD and ISSR primers. Their data showed our soybean isolates RESULTS The baiting bioassay detected Phytophthora in naturally infested soybean soils from Iran. These baits differed significantly in detection of Phytophthora and in contamination by other microorganisms. The percentage of soybean leaf disks detecting Phytophthora was consistently the highest and usually was significantly greater than those for the other baits tested. Duration of baiting (i.e., the period when baits were floated over flooded container mixes) affected both detection of Phytophthora and incidence of contaminants. Significantly more baits detected Phytophthora at 24 h than at 6 or 12 h; however, 24 h-baits also had more contamination than 6and 12 h-baits. The temperature during baiting did not have a dramatic effect on the detection of Phytophthora or incidence of contamination. Predominant isolates belonged to P. sojae. Ten isolates were obtained belonging to P. nicotianae. The phenotypic characteristics of these isolates were fluffy cottony mycelium with slightly striated pattern; growth rate on the PDA was 5 mm per day. None of the isolates grew at 5 °C but they showed growth at 35 °C. Sporangia were produced abundantly in sterile soil extract; they were papillate, predominantly ovoid or obpyriform. All isolates formed sexual structures when paired with the opposite mating type. They showed spherical and smooth oogonia with amphigynous antheridia. All isolates obtained from soybean plants belonged to the mating type A2. All isolates showed spherical, aplerotic oospores, which ranged from 27.5 to 28.8 m in diameter and with an oospore wall of 2.0 to 2.2 m thick. Soybean seedlings died five days after inoculation. Pathogenicity tests showed that the isolates were pathogenic on soybean seedling. P. nicotianae isolates were sent to another research group for test by ITS primers. Their results proved P. nicotianae. DISCUSSION A number of techniques are used for the detection of multiple species of Phytophthora within a Journal of Agrobiology, 29(1): 29­32, 2012 were separated at 75% as with other Iranian isolates (Ranjbaran et al. 2006). O'Brien PA, Williams N, Hardy GES (2009): Detecting Phytophthora. Critical Reviews in Microbiology 35: 169­181. Parkunan V, Johnson C, Bowman B, Hong C (2010): Population structure, mating type, and mefenoxam sensitivity of Phytophthora nicotianae in Virginia tobacco fields. Plant Disease 94: 1361­1365. Ranjbaran M, Alizadeh A, Safaie N (2006): Genetic diversity of Iranian populations of Phytophthora nicotianae using ISSR and RAPD markers. Iranian Journal of Plant Pathology 42: 619­638. Reeser PW, Sutton W, Hansen EM, Remigi P, Adams GC (2011): Phytophthora species in forest streams in Oregon and Alaska. Mycologia 103: 22­35. Tsao PH (1983): Factors affecting isolation and quantitation of Phytophthora from soil. In Erwin DC, Bartnicky Garcia S, Tsao PH (eds.). Phytophthora its Biology, Taxonomy, Ecology, and Pathology. St. Paul, Minnesota: American Phytopathological Society (APS Press), pp. 219­236. Wu X, Zhou B, Zhao J, Guo N, Zhang B, Yang F, Chen S, Gai J, Xing H (2011): Identification of quantitative trait loci for partial resistance to Phytophthora sojae in soybean. Plant Breed 130: 144­149. Yamak F, Peever TL, Grove GG, Boal RJ (2002): Occurrence and identification of Phytophthora spp. pathogenic to pear fruit in irrigation water in the Wenatchee River Valley of Washington state. Phytopathology 92: 1210­1217. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Agrobiology de Gruyter

A simple method for detection of Phytophthora nicotianae from soybean soil

Mohammadi, Abbas
Journal of Agrobiology , Volume 29 (1) – Jan 1, 2012
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de Gruyter
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Copyright © 2012 by the
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1803-4403
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1804-2686
DOI
10.2478/v10146-012-0004-4
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Abstract

A high detection rate of Phytophthora nicotianae was obtained from the naturally infested soil of soybean fields. Air-dried soils were firstly moistened in a flask and then pre-incubated at 25 °C for 1­4 weeks before flooding with distilled water and baiting with soybean leaf disks for 6, 12, and 24 h. The baits were then thoroughly washed, flooded with 10­15 ml of distilled water in Petri-dishes and incubated under continuous fluorescent light for 72 h. Sporangia started to emerge from the margins of leaf disks. They were easily observed under the stereomicroscope. Pure culture of the fungus was obtained by spreading zoospore suspension on a 1.5% water-agar containing anti-bacterial antibiotics. This is the first report of recovery of P. nicotianae from naturally infested soybean soils using sensitive leaf disks of soybean (Williams cultivar) as bait in Iran. All isolates were determined to be A2 mating type. Key words: Phytophthora; Glycine max; detection; baiting technique; mating type INTRODUCTION Baiting techniques use plant material to isolate selectively the pathogen from soil or diseased roots. The baiting technique is commonly used for detection of Phytophthora occurrence in soils (Erwin and Ribeiro 1996, Mohammadi et al. Abbas Mohammadi, Department of Plant Pathology, University of Birjand, Birjand, Iran Amohammadi@birjand.ac.ir 2008). The technique involves floating pieces of susceptible tissue on a soil water slurry with a high water/soil ratio (Erwin and Ribeiro 1996). Zoospores formed by Phytophthora in the sample infect the baits which can be detected by plating baiting tissue onto selective agar containing antibacterial and antifungal antibiotics allowing outgrowth of Phytophthora from the tissue (Reeser et al. 2011). Phytophthora species growing out of the bait can be identified on the basis of colony morphology, mycelial characteristics, cardinal growth temperatures, and production, morphology, and dimensions of sporangia, oogonia, and antheridia, or DNA sequence analysis (Lamour and Kamoun 2009). Depending 29 Journal of Agrobiology, 29(1): 29­32, 2012 on the time of year at which the sample is taken, the efficiency of detection ranges from 0 to more than 90%. The host species from which the bait tissue is derived also influences the efficiency of detection (O'Brien et al. 2009). Although the bait tissue is derived from a host species that is susceptible to the pathogen, different host species give very different efficiencies of detection (Erwin and Ribeiro 1996). Isolation of Phytophthora from the infected bait requires a considerable time, the use of selective media and considerable knowledge of the genus(Yamak et al. 2002). A major problem is the presence of fast growing organisms such as Pythium, which tend to inhibit growth of the target species (Canaday and Schmitthenner 1982). The infection of the bait can be a limiting factor. The efficiency of detection of Phytophthora in soil by the baiting technique can be improved by drying out the soil and rewetting it (double baiting demonstrating that, as with tissue sections, the pathogen, although present, will not always grow out of the sample (Canaday and Schmitthenner 1982). Davison and Tay (2005) found that double baiting increased the recovery of positive samples. The quality of the water used can also significantly affect the outcome as zoospores are very sensitive to toxic ions present in unpurified water (Tsao 1983). The objectives of this study were to develop a rapid and sensitive baiting method for isolation of P. nicotianae from the soil carried with transported soybeans or in production fields, and to develop a rapid assay to detect the pathogen in infected soybean fields. were floated immediately on the surface of the water. After 6, 12, and 24 h, the floating leaf disks were removed, washed, and placed in another Petri dish with 25 ml of sterile distilled water. Sporangia emerging from the edge of the infected leaf discs were observed under a stereomicroscope after 48 h of incubation in the water. Leaf discs with sporangia of Phytophthora were placed in another Petri dish with 10 ml of sterile distilled water. The entire set of Petri dishes was placed in a refrigerator at 4 °C for 30 min and then placed back at 25 °C for 1 h to complete the process of zoospore release. The zoospore solution was spread on 1.5% water agar containing pentachloronitrobenzene at 25 g ml­1, carbendazim at 25 g ml­1, rifampicin at 20 g ml­1, and ampicillin at 50 g ml­1. The germinated zoospores were isolated 12 h later and pure cultures were obtained (Erwin and Ribeiro 1996, Hua-Bo and Xiao-Ming 2003). Cultures were grown on 5% clarified carrot agar and maintained with regular transfers. Vegetative and reproductive stages were examined in cultures grown for 1 to 4 weeks. Isolates were scored for appearance of hyphae, size, and shape of sporangia and chlamydospores, and the presence or absence of oospores. Observations were compared to published descriptions for Phytophthora species (Ho 1981, Erwin and Ribeiro 1996, Ranjbaran et al. 2006, Gallegly and Hong 2008). The mating types of isolates were determined by pairing known A1 and A2 testers of P. nicotianae on 10% clarified V8 juice agar medium. The unknown isolate was placed on one side of the 60-mm-diameter Petri dish and the known A1 or A2 tester was placed on the other side. The Petri dishes were incubated in the dark at 25 °C for 4 to 8 weeks or until oospores were formed. The mating type was identified for unknown isolates based on the presence of oospores. If pairing with the known A1 tester produced oospores, then the unknown isolate was determined to be A2, and vice versa. If isolates did not produce oospores after 8 weeks, a second attempt was made using previously identified P. nicotianae testers (Ranjbaran et al. 2006). Inoculums for pathogenicity tests were prepared by growing isolates on oatmeal agar in Petri plates at 25 °C for 2 weeks. Inoculations were performed by the standard hypocotyls method (Mohammadi et al. 2008), using 2 × 2 mm pieces of mycelia, and the wound was covered to prevent desiccation of the inoculums and host tissue. Ten 7-day-old seedlings of soybean MATERIALS AND METHODS Soil samples were collected from soybean fields from Golestan, Mazandaran, and Lorestan provinces, Iran, during growing seasons. Five individual samples were taken arbitrarily to a depth of 15 cm using a hand trowel and were well mixed to represent a single composite sample for each field, with a final volume of approximately 100 cm3. All soil samples were air dried, triturated, and then stored in paper pockets at room temperature. P. nicotianae isolation from soil was made by modification of the soybean leaf baiting method (Canaday and Schmitthenner 1982, Hua-Bo and Xiao-Ming 2003). Soil samples were moistened, preincubated at 25 °C for 1­4 weeks, and then flooded with sterile distilled water. Twenty leaf disks from the Williams soybean seedlings Journal of Agrobiology, 29(1): 29­32, 2012 were inoculated with each isolate and grown in the greenhouse at 25±2 °C with supplemental fluorescent and incandescent light (Mohammadi et al. 2008, Wu et al. 2011). sample (Gallegly and Hong 2008). Soybean leaf disk is a specific baiting method to P. sojae (Canaday and Schmitthenner 1982). Despite this host specificity, these baits also attract Pythium species. This study showed that soybean leaf disks baiting were effective for isolating P. nicotianae from soybean soils. Prewetting of soil for various periods of time before submersion in water favours the production of sporangia. Other studies showed that prewetting soil before dilution plating or baiting with pepper leaf disks favoured the detection of oospore inoculums of P. capsici. Double baiting increased the recovery of positive samples from 1.9 to 2.5% and 6.3 to 7.5% of samples taken from the centre and margins respectively of disease fronts in Western Australia (Davison and Tay 2005). When leaf disks are floated for a short time (about 1 h), P. sojae is more frequently detected than other fungi, but at longer incubation times (12 h), P. nicotianae is also detected. At incubation times longer than 24 h, Pythium species are also detected on leaf disks. Ten isolates from Phytophthora species that were detected from infected soybean soils from Iran were P. nicotianae. This is the first record of P. nicotianae on soybean in Iran. This data showed that P. sojae is the major causal agent of soybean stem rot in Iran (Mohammadi et al. 2008). The second species is P. nicotianae. All isolates were detected as being the A2 mating type. Ranjbaran et al. (2006) showed 95% of P. nicotianae from Iran were the A2 mating type. Information on distribution of A1 and A2 mating types is critical to determine the genetic variability within the field pathogen population. Only one-tenth of the Virginia tobacco fields had both mating types, and all those samples originated from fluecured tobacco fields, indicating a low genetic diversity (Parkunan et al. 2010). The relative ratios of A1 and A2 mating types could suggest the level of sexual recombination occurring in natural field conditions, as well as the potential for oospore production. Predominance of a single mating type in the majority of Iran soybean fields may indicate genetically less diverse populations (Ranjbaran et al. 2006). P. nicotianae isolates were sent to another research group in the Tarbiat Modares University in Iran. Their results with specific primers (par A1) were in agreement with our morphological observation and detected them as P. nicotianae. They studied one isolate from California and 25 isolates from twenty hosts by ten RAPD and ISSR primers. Their data showed our soybean isolates RESULTS The baiting bioassay detected Phytophthora in naturally infested soybean soils from Iran. These baits differed significantly in detection of Phytophthora and in contamination by other microorganisms. The percentage of soybean leaf disks detecting Phytophthora was consistently the highest and usually was significantly greater than those for the other baits tested. Duration of baiting (i.e., the period when baits were floated over flooded container mixes) affected both detection of Phytophthora and incidence of contaminants. Significantly more baits detected Phytophthora at 24 h than at 6 or 12 h; however, 24 h-baits also had more contamination than 6and 12 h-baits. The temperature during baiting did not have a dramatic effect on the detection of Phytophthora or incidence of contamination. Predominant isolates belonged to P. sojae. Ten isolates were obtained belonging to P. nicotianae. The phenotypic characteristics of these isolates were fluffy cottony mycelium with slightly striated pattern; growth rate on the PDA was 5 mm per day. None of the isolates grew at 5 °C but they showed growth at 35 °C. Sporangia were produced abundantly in sterile soil extract; they were papillate, predominantly ovoid or obpyriform. All isolates formed sexual structures when paired with the opposite mating type. They showed spherical and smooth oogonia with amphigynous antheridia. All isolates obtained from soybean plants belonged to the mating type A2. All isolates showed spherical, aplerotic oospores, which ranged from 27.5 to 28.8 m in diameter and with an oospore wall of 2.0 to 2.2 m thick. Soybean seedlings died five days after inoculation. Pathogenicity tests showed that the isolates were pathogenic on soybean seedling. P. nicotianae isolates were sent to another research group for test by ITS primers. Their results proved P. nicotianae. DISCUSSION A number of techniques are used for the detection of multiple species of Phytophthora within a Journal of Agrobiology, 29(1): 29­32, 2012 were separated at 75% as with other Iranian isolates (Ranjbaran et al. 2006). O'Brien PA, Williams N, Hardy GES (2009): Detecting Phytophthora. Critical Reviews in Microbiology 35: 169­181. Parkunan V, Johnson C, Bowman B, Hong C (2010): Population structure, mating type, and mefenoxam sensitivity of Phytophthora nicotianae in Virginia tobacco fields. Plant Disease 94: 1361­1365. Ranjbaran M, Alizadeh A, Safaie N (2006): Genetic diversity of Iranian populations of Phytophthora nicotianae using ISSR and RAPD markers. Iranian Journal of Plant Pathology 42: 619­638. Reeser PW, Sutton W, Hansen EM, Remigi P, Adams GC (2011): Phytophthora species in forest streams in Oregon and Alaska. Mycologia 103: 22­35. Tsao PH (1983): Factors affecting isolation and quantitation of Phytophthora from soil. In Erwin DC, Bartnicky Garcia S, Tsao PH (eds.). Phytophthora its Biology, Taxonomy, Ecology, and Pathology. St. Paul, Minnesota: American Phytopathological Society (APS Press), pp. 219­236. Wu X, Zhou B, Zhao J, Guo N, Zhang B, Yang F, Chen S, Gai J, Xing H (2011): Identification of quantitative trait loci for partial resistance to Phytophthora sojae in soybean. Plant Breed 130: 144­149. Yamak F, Peever TL, Grove GG, Boal RJ (2002): Occurrence and identification of Phytophthora spp. pathogenic to pear fruit in irrigation water in the Wenatchee River Valley of Washington state. Phytopathology 92: 1210­1217.

Journal

Journal of Agrobiology – de Gruyter

Published: Jan 1, 2012

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