Final Report for GNC02-006
Project Information
Forty-five species of crop and weed plants were screened for their susceptibility to P. capsici. Twenty-two crop species succumbed to the disease, 14 did not. Pathogenicity tests in the greenhouse showed that Phytophthora capsici isolates were significantly less virulent on eggplant than they were on cucurbits, pepper, and tomato. Molecular study showed that there are significant differences in pathogenicity and genetics among isolates of P. capsici. The results of this research will help in establishing effective cropping rotations for management of P. capsici in vegetable and weed control, thus establishing sustainable vegetable, particularly cucurbit, production.
Introduction:
Illinois farmers grow approximately 12,000 acres of jack-o-lantern pumpkins and 10,000 acres of processing pumpkins each year (3). About 90% of commercial processing pumpkins produced in the United State (US) are grown in Illinois. Also about 10,000 acres of cantaloupe, cucumbers, eggplants, pepper, tomatoes, and watermelon are grown in Illinois each year. Phytophthora blight, caused by Phytophthora capsici Leonian, has become one of the most serious threats to production of cucurbits, eggplants, and peppers in Illinois, as well as nationwide (3,5,8,23,24). Phytophthora blight causes up to 100% yield losses in commercial fields of the above-mentioned crops in Illinois (2,3). Heavy crop losses of cucurbits, particularly pumpkins, to Phytophthora blight, lack of resistant cultivars, and inadequate effect of chemicals in controlling the disease prompted our investigation to develop strategies to manage the disease and minimize crop losses. Crop rotation to minimize primary inoculum of P. capsici in infested fields is an important component of disease management strategies.
Phytophthora capsici is a soil-borne Oomycete and survives as oospores in soil for several years (5,29). The pathogen can infect all parts of the plant at any growth stage, causing seedling death, crown rot, leaf spot, foliar blight, and fruit rot (3,5,29). Infection of the foliage occurs when zoospores of P. capsici are splashed onto the plant surfaces from soil during rainfall or irrigation (16). Propagules of P. capsici are dispersed by water, soil, and air currents (22). Seedling death occurs in wet and warm (20 to 30C) soil conditions (5,11). Therefore, screening seedlings for their susceptibility to P. capsici in a greenhouse is a reliable approach for determining host range of P. capsici and disease resistance.
Forty-nine plant species have been reported infected with P. capsici (3). Among the major hosts of P. capsici are peppers (Capsicum annuum), watermelon (Citrullum lanatus), cantaloupe (Cucumis melo), honeydew melon (C. melo), cucumber (Cucumis sativus), blue Hubbard squash (Cucurbita maxima), acorn squash (Cucurbita moschata), gourd (C. moschata), processing pumpkin (C. moschata), yellow squash (C. pepo), zucchini squash (C. pepo), tomato (Lycopersicon esculentum), black pepper (Piper nigrum), and eggplant (Solanum melongena).
Cucurbit isolates of P. capsici have been reported pathogenic on cucurbits, pepper, and tomato (13). Polach and Wenster (20) reported distinct pathogenic strains identified among isolates of P. capsici from tomato, pepper and squash. Ristaino (23) evaluated the relative virulence of isolates of P. capsici from cucurbits (cucumber and squash) on pepper and found differences in virulence among the isolates. In Italy, Tamietti and Valentino (24) grouped P. capsici isolates into 13 classes depending on their ability to infect different plant species (pepper, tomato, eggplant, melon, squash, pea, and French bean). In South Korea, Lee et al. (16) studied aggressiveness of P. capsici isolates from pepper and pumpkin on pumpkin cultivars and reported significant pathogen-host interactions.
Visual observations of symptoms and isolation of the pathogen from infected tissue have been the methods employed for diagnosing diseases caused by P. capsici (3,17). However, this approach is laborious and time consuming. Therefore, it was necessary to develop a rapid and sensitive diagnostic method for detection of P. capsici in plant tissue. The polymerase chain reaction (PCR) assay is an approach that allows for the rapid detection of Phytophthora species in plants (16,22,24,25).
Genetic diversity is common among isolates of fungal species (14,27). Different methods have been used to study the genetic variation of fungi (8,22,24,28). Internal transcribed spacers (ITS) regions have been used to determine genetic differences among species of Phytophthora, as well as other fungi (22,28). Inter-simple sequence repeats (ISSR) amplification is a new technique that could rapidly differentiate closely related individuals within a fungal species (28). Amplified fragment-length polymorphism (AFLP) is a recently developed polymerase chain reaction (PCR) that provides genetic markers for fingerprinting, mapping, and studying genetic relationships among populations within fungal species (12,21). Alonoso & Glenn (1) modified the original AFLP and provided an easy protocol for digestion of genomic DNA and ligation with adapters in one reaction.
Genetic variation among P. capsici isolates has been reported in some vegetable growing areas in the world (9,18). Our research reports the results of investigation on pathogenic and genetic variation among P. capsici isolates on eggplant, pepper, pumpkin, squash, tomato, and watermelon in Illinois.
References
1. Alonso S, Glenn HH, 1999. Modification of the AFLP protocol applied to honey bee DNA. Biotechnology 26, 706-709.
2. Babadoost, M. 2000. Outbreak of Phytophthora foliar blight and fruit rot in processing pumpkin fields in Illinois. Plant Dis. 84: 1345.
3. Babadoost, M., and Islam, S.Z. 2003. Fungicide seed treatment effects on seedling damping-off of pumpkin caused by Phytophthora capsici. Plant Dis. 87: 63-68.
4. C.M.I. 1985. C.M.I. description of pathogenic fungi and bacteria, No, 836. Phytophthora capsici. CAB, Kew, England.
5. Erwin, D.C., and Ribeiro, O.K. 1996. Phytophthora Diseases Worldwide. American Phytopathological Society Press, St. Paul, MN.
6. Farr, D.F., Bills, G.F., Chamuris, G.P., and Rossman, A.Y. 1995. Fungi on Plants and Plant products in the United States. American Phytopathological Society, St. Paul, MN.
7. Forster H, Odemans P, Coffey MD, 1990. Mitochondrial and nuclear DNA diversity within six species of Phytophthora. Exp. Mycol. 14, 18-31.
8. Hwang BK, Arthur WA, Heitefuss R, 1991. Restriction fragment length polymorphisms of mitochondrial DNA among Phytophthora capsici isolates from pepper (Capsicumannuum). System. Appl. Micriobiol. 14, 111-116.
9. Hwang, B.K., and Kim, C.H. 1995. Phytophthora blight of pepper and its control in Korea. Plant Dis. 79: 221-227.
10. Innis MA, Gelfand DH, Sninsky JJ, White TJ, 1990. PCR protocols: a guide to methods and applications. Academic Press, Inc., New York, N.Y.
11. Islam, S.Z., and Babadoost, M. 2002. Effect of red-light treatment of seedlings of pepper, pumpkin, and tomato on the occurrence of Phytophthora damping-off. HortSci. 37: 678-681.
12. Janssen PR, Coopman G, and Swings HJ 1996. Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy. Microbiology 142, 1881-1893.
13. Kreutzer WA, Bodine EW, Durrell LW, 1940. Cucurbit diseases and rot of tomato fruit caused by Phytophthora capsici. Phytopathology 30, 972-976.
14. Lamour, K.H., and Hausbeck, M.K. 2002. The spatiotemporal genetic structure of Phytophthora capsici in Michigan and implications for disease management. Phytopathology 92: 681-684.
15.Lantin, R.X., and Rane, K. 1999. Identification and Management of Pumpkin Diseases. BP-17, Purdue University, Lafayette, IN.
16. Lee, B.K., Kim, B.S., Chang, S.W., and Hwang, B.K. 2001. Aggressiveness to pumpkin cultivars of isolates of Phytophthora capsici from pumpkin and pepper. Plant Dis. 85: 497-500.
17. Leonian, L.H. 1922. Stem and fruit blight of peppers caused by Phytophthora capsici. Phytopathology 12: 401-408.
18. Majer DR, Mithen BG, Lewis PV, Oliver RP, 1996. The use of AFLP fingerprinting for the detection of genetic variation in fungi. Mycol. Res.100, 1107-1111.
19.Papavizas GS,, Bowers JH, Johnston SA, 1981. Selective isolation of Phytophthora capsici form soil. Phytopathology 71, 129-133.
20. Polach FJ, Wenster RK, 1972. Identification of strains and inheritance of pathogencity in P. capsici. Phytopathology , 20-26.
21. Questiau S, Eybert M, Taberlet P, 1999. Amplified fragment length polymorphism(AFLP) markers reveal extra-pair parentage in a bird species: The blue throat (Lucinia svecica). Mol. Ecol. 8, 1331-1339.
22. Ristaino JB, Parra G, 1998. PCR Amplification of ribosomal DNA for species identification in the plant pathogen genus Phytophthora. Appl. Environ. Microbiol. 64, 948-954.
23. Ristaino, J.B., and Johnston, S.B. 1999. Ecologically-based approaches to management of Phytophthora blight on bell pepper. Plant Dis. 83: 1080-1089.
24. Tamietti, G. and Valentino, D. 2001. Physiological characterization of a population of Phytophthora capsici Leon. from northern Italy. J. Plant Pathol. 83: 1101.
25. Tooley, P.W., Bunyard, B.A., and Hatziloukas, E. 1997. Development of PCR primers from internal transcribed spacer region II for detection of Phytophthora species infecting potatoes. Appl. Environ. Micriobiol. 63: 1467-1475.
26. Trout, C.L., Ristaino, J.B., Madritch, M., and Wandsomeboondee, T. 1997. Rapid detection of Phytophthora infestans in late blight-infected potato and tomato using PCR. Plant. Dis. 81: 1042-1048.
27. Zhou, Z., Miwa, M., and Hogetsu, T. 2001. Polymorphism of simple sequence repeats reveals gene flow within and between ectomycorrhizal Suillum grevillei populations. New Phytologist. 149: 339-349.
28. White TJ, Bruns T, Lee S, Taylor J, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages315-322 in: PCR protocols: a guide to methods and applications. Innis MA, Gelfand DH, Sninsky JJ, and White TJ (ed.). Academic Press, Inc., New York.
29. Zitter, T.A., Hopkins, D.L., and Thomas, C.E. 1996. Compendium of Cucurbit Diseases. American Phytopathological Society, St. Paul, MN.
The main objective of this study was to determine pathogenic diversity of P. capsici isolates for establishing sustainable pumpkin production in Illinois. The specific objectives of this research were:
1. Determine the susceptibility of crops grown in rotation with cucurbits and weeds that commonly grow in cucurbit fields to P. capsici.
2. Assess the virulence of P. capsici isolates on different pumpkin cultivars.
3. Determine genetic and pathogenicity variation among isolates of P. capsici from Illinois.
Cooperators
Research
Phytophthora capsici was isolated from infected plant tissues by culturing diseased tissue onto a semi-selective medium (PARP) (11,19). The isolates were maintained on lima bean agar (LBA, Difco Lab., Detroit, MI). Twenty-two isolates of P. capsici from Illinois and two from China were used in this study (Table 1). All isolates were tested to determine their mating types by pairing with known A1 (ATCC-15427) or A2 (ATCC-15399) tester isolates of P. capsici that were obtained from the American Type Culture Collection (ATCC).
Virulence test. Three processing pumpkin cultivars (Dickinson, H-401, H-608) and three jack-o-lantern pumpkin cultivars (Gold Rush, Gold Medal, and Pik-A-Pie) were used to determine the virulence of P. capsici isolates of pumpkins. Six isolates of P. capsici, three A1 and three A2 mating types from processing pumpkins were used in this experiment (Table 2). Sporangial suspensions were prepared from 5-day-old culture plates of P. capsici grown on LBA at 24C under continuous white fluorescent light. Ten milliliters of sterile distilled water (SDW) was added to each plate and the sporangia were dislodged using a soft brush. Sporangial suspensions from six isolates (equal numbers of plates of each isolate) were mixed. The suspension was then incubated at 20C for 1h to allow the sporangia to release their zoospores. Zoospores were separated from the empty sporangia by passing the suspension through a four-layer facial tissue. The concentration of zoospores was 2 ×105 zoospores per ml of water. Seeds were planted in 10-cm-diameter pots containing a soil:sand:vermiculite mix and grown in the greenhouse. Four-week-old seedlings were inoculated by adding 5 ml of the zoospore suspension to each pot as described above. Control plants received 5 ml of SDW. Beginning the second day after inoculation, plants were evaluated for disease incidence until 21 days after inoculation. Disease incidence was assessed as percentage of seedlings that died. Area under disease progress curve (AUDPC) was calculated using the formula: AUDPC = n Σ i = 1 (Xi + 1 + Xi) (ti + 1 – ti)/2, where Xi= disease incidence at the ith observation, ti= days at the ith observation, and n = total number of observations. The experiment was performed using a randomized complete block design with four replications each with 10 plants. The experiment was conducted twice.
Host range. Forty-five species of plants were screened for their susceptibility to P. capsici (Table 3). Sixteen soybean cultivars (Bell, Harosoy13, Harosoy 16, Harodoy 63, L75-3735, L76-1988, L83-570, L85-2352, L85-3059, L89-1581, L93-3258, Resink, Saloan, Williams, Williams 82, Union) were included in this test. Seeds of the plants were sown in 10-cm-diameter plastic pots (one seed per pot) containing steamed soil mix (soil: sand: vermiculite; 1:1:1) and were grown on a greenhouse bench at 18 to 26C. Four-week-old seedlings were inoculated by applying a suspension of P. capsici zoospores over the soil surface around plants in each pot (5 ml of 2 ×105 spores/seedling/pot). Control seedlings received 5 ml of SDW. Seedlings were watered before inoculation to keep the soil wet. After inoculation, the pots were placed in plastic trays containing water that kept the soil moist for at least 12 h. The seedlings were then placed on the greenhouse bench and watered twice daily. At the beginning of the second day after inoculation, seedlings were evaluated for the development of lesions on stems, defoliation and damping-off symptoms, every day for three weeks. The experiment was performed using a randomized complete block design with four replications each with 10 plants. The experiment was repeated twice.
Pathogenicity test. Six plant species, including pumpkin (Cucurbita pepo, ‘Gold medal’), squash (Cucurbita pepo, ‘Sebring F1’), tomato (Lycopersicon esculentum, ‘Popreco’), pepper (Capsicum annum, ‘California Wonder’), eggplant (Solanum melongena, ‘Classic’), and watermelon (Citrullum lanatus, ‘SWT6703’) were used in this experiment. Twenty-four isolates were used to inoculate the plant. The inoculation method, data recording and analysis were the same as above.
Molecular study. The single spore isolates of P. capsici were grown in liquid lima bean broth for six days. Fresh mycelium was harvested by filtration. Samples were frozen in liquid nitrogen and ground to a fine powder. Total genomic DNA was extracted from mycelium by using the CTAB procedure (10).
ITS regions were amplified using two primers ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG) and ITS4 (5’-TCCTCCG CTTATTGATATGC). Each reaction contained 20 ng DNA, 1x PCR buffer, 200 μM d NTP, 2.0 μM MgCl2, 0.5 μM forward and reverse primers each, and 5 U Tag DNA polymerase. DNA amplification was performed in a thermal cycler (PTC-200, MJ Research Inc., Waltham, MA) with one cycle at 94C for 3 min, followed by 35 cycles consisting of 94C for 1 min, 50C for 1 min, extension at 72C for 1 min, and a final extension at 72C for 10 min. Fifteen restriction enzymes (HifI, HaeIII, PstI, SstI, SspI, TaqI, AccI, NccI, SpeI, BgtII, ClaI, RsaI, XhoI, AvaII, and MboI) were used to digest the product of PCR-amplified ITS regions. Restriction digests were carried out using 10 μl of PCR product in 15 μl reaction mixtures at 37C for 2 h. Phytophthora sojae was used as a control.
The ISSR-PCR was performed in 25 μl volume, including 40 ng template DNA, 1X PCR buffer, 200 μM d NTP, 2.5 μM MgCl2, 40 nM primer (Table 3), and 0.8 U Tag DNA polymerase. The PCR reactions were performed using the PTC 200 Thermal Cycler. The PCR test consisted of one cycle at 95C for 2 min, followed by 35 cycles consisting of 94C for 1 min, annealing for 1 min (temperature was different for different primers), extension at 72C for 1 min, and a final extension at 72C for 10 min. To select appropriate primers for amplification in the ISSR test, 20 primers were tested (Table 3).
A modified AFLP procedure, developed by Alonso and Glenn (1), was also used in this study to compare genetic variation of the P. capsici isolates. Same isolates used in the ISSR tests (Table 1) were used in AFLP tests. Two micrograms of genomic DNA were added to 100 μl of 25 mM Tris-HAc with pH of 7.8, 10 mM MgAc, 5 mM KAc, 5 mM dithiotreitol (DTT), 50 ng/μl bovine serum albumin (BSA), and 1 mM ATP. Twenty units of EcoRI, 1 U of T4 ligase, and 50 pmol of the EcoRI adapter were added, and incubated at 37C overnight. By using 0.1 volumes of 3 M NaOAc and 3 volumes of cold 100% ethanol, unincorporated adapters and residual enzymes were precipitated, removed, and re-suspended in 100 μl TE buffer. The PCR test was performed in 25 μl total volume, including 1 μl digest-ligation product, 1 X PCR buffer, 200 μM d NTP, 2.5 μM MgCl2, 40 nM primer and 0.8 U Tag DNA polymerase. The PCR reactions were performed on the PTC-200 Thermal Cycler. The PCR profile consisted of one step at 95C for 2 min, followed by 35 cycles consisting of 94C for 40 sec, 58C for 1min, extension at 72C for 1 min, and a final extension at 72C for 10 min.
Products of ITS-digestion and ISSR-PCR and AFLP-PCR tests were resolved in a 1.5% agarose-SynergelTM (0.7% agarose and 0.4% Synergel; Diversified Biotech, Boston, MA) in 1 X TBE buffer and electrophoresed. Gel tests were run at 60 V for 4 h, stained with ethidium bromide and viewed under UV light to visualize products.
Data analysis. Analysis of variance procedures of SAS (SAS Institute, Cary, NC) were used to compare AUDPCs and host pathogen interactions. Since there was no significant difference in AUDPC values between the experiments, the results of two experiments were combined and presented. Comparison of amplified DNA profiles for each of the primers was performed on the basis of the presence or absence of ISSR and AFLP fragments. The presence or absence of amplified DNA fragments was determined for each isolate and scored as 1 or 0, respectively. Cluster analysis was based on the unweighted paired-group method using arithmetic averages (UPGMA). Bootstrapping with 1000 replications was carried out and a majority rule consensus tree was generated with the Mega 2.1 program.
Virulence test. The relative virulence of six isolates of P. capsici on six pumpkin cultivars was evaluated by comparing percentage of plant death. Percentage of plant death was significantly affected by pathogen isolate and pumpkin type × isolate interactions (Table 5). There was no significant effect of pumpkin cultivar on percentage of plant death. There was significant difference in percentage of seedling death between jack-lantern and processing pumpkins (Table 6). The standard deviations in percentage of plant death for jack-o-lantern and processing pumpkin cultivars were 38.27 and 34.28.
We found that isolates of P. capsici differ in virulence, which agrees with the reports by previous investigators (9,16,22). The isolates used in this study were less virulent on processing pumpkins than on jack-o-lantern pumpkins. Therefore, more effective measures are needed to manage P. capsici in jack-o-lantern pumpkin fields. None of the pumpkin cultivars used in this study showed resistance to P. capsici. This may be another indication that there is no measurable resistance in pumpkin cultivars to P. capsici, as reported by Erwin (5) and Lantin (15). Consequently, the effectiveness of other methods (e.g., cultural practice, chemical treatments and induced resistance) should be investigated to develop effective integrated strategies for management of P. capsici in pumpkin fields.
Host range. Plants of 22 crop species and two weed species exhibited damping-off symptoms (Table 4). Plants of 14 crop species and seven weed species did not develop any symptom. All plants from Cucurbitaceae, Solanaceae, and Chenopodiaceae families became infected and developed symptoms. Cucurbits and pepper were the most susceptible to P. capsici, as more than 50% and more than 95% of seedlings became infected and developed symptoms within 3 and 12 days after inoculation, respectively. Infection in eggplant, tomato, tobacco, nightshade, spinach, beet, Swiss chard, green bean, lima bean, snow pea, turnip, radish, carrot, and velvetleaf developed symptoms slowly. However, more than 50% of seedlings became infected and developed symptoms within 10 days from inoculation. Onion was less susceptible and only 41.9% of seedlings exhibited symptoms. No obvious changes in symptom development were observed after 12 days post inoculation. P. capsici was re-isolated from all of the symptomatic plants. None of the control plants developed disease symptoms, and attempts to isolate P. capsici from their tissues were unsuccessful. Thus, the results of control plants are not presented.
Basil, broccoli, cabbage, cauliflower, celery, chive, corn, dill, kale, kohlrabi, mustard, parsley, soybean, and wheat seedlings didn’t develop any symptoms. Likewise, the weed species of cocklebur, crab grass, lamb’s-quarters, pigweed, puncture vine, sandbur, and water hemp did not develop symptoms. P. capsici from asymptomatic plant tissues of inoculated plants did not provide any indication of presence of P. capsici in these plants.
The results of this study agree with the reports by other investigators (5,15,16,20,26) that cucurbits and peppers are the most susceptible hosts of P. capsici. The lists of P. capsici hosts have been published by other investigators (4,5,6). The list assembled by Erwin and Ribeiro is the most comprehensive list of P. capsici hosts worldwide. They list 49 species of herbaceous and woody plants can be infected by P. capsici. We tested crops that are used in rotation sequences of pumpkin in Illinois and found that most of the plant species that have previously reported as hosts of P. capsici (4,5,6) can be infected by isolates of this pathogen from pumpkin in Illinois. However, cauliflower (Brassica oleracea var. botrytis), listed as a host of P. capsici (5,22), was not infected by isolates of P. capsici from pumpkin in Illinois (Table 4). This indicates that either cauliflower is not a host of P. capsici or it is resistant at early growth stages and may become susceptible as the plant matures.
This is the first report of beet (Beta vulgaris), Swiss chard (Beta vulgaris var. cicla), lima bean (Phaseolus lunatus), turnip (Brassica rapa), spinach (Spinacia olerace) and velvetleaf (Abutilon theophrasti) as hosts of P. capsici. Wild spinach (Chenopodium amaranticolor), however, has been previously reported as a host of P. capsici (5,23). Nightshade and velvetleaf are weeds that commonly grow in commercial fields of pumpkins and other cucurbits. Soybean, corn, and wheat, the major crops grown in Illinois, were not infected with P. capsici in our study, and there is no report indicating that these crops could be infected with P. capsici. This report is expected to help in establishing appropriate rotations and weed management programs for sustainable pumpkin production.
Since inoculation tests were conducted under conditions highly conducive for disease development (tender greenhouse-grown seedlings and high inoculum dose), it is possible that some of the species susceptible to P. capsici in the greenhouse may not be as susceptible under field conditions. Field studies could provide additional information on the susceptibility of the species tested in the greenhouse in this study.
Pathogenicity test. All isolates of P. capsici were pathogenic on six plant species (pumpkin, squash, watermelon, eggplant, pepper, and tomato) tested. Inoculated plants developed symptoms. No symptoms were observed on control plants. Area under developing progress curve (AUDPC) values (percentage of seedlings died) on six hosts differed significantly (P = 0.05) from each other (Table 7). Cucurbits (pumpkin, squash, watermelon) were more susceptible than other three plant species to P. capsici. The AUDPC values on eggplant were significantly lower than those of other hosts.
Both A1 and A2 mating types of P. capsici were identified in both central and southern Illinois. All of the isolates produced oospores when paired with a compatible mating type. The presence of both of the mating types in the area may increase the potential of genetic recombination in P. capsici and appearance of new pathogenic strains.
Isolates of P. capsici, which were collected from different locations exhibited variation in pathogenicity on different hosts. Disease incidence on cucurbits was significantly higher than those of eggplant, pepper, and tomato, and disease incidence on eggplant was significantly lower than those of other plants. These results agree with the reports by Ristaino (22) and Lee et al. (16) indicating significant differences in aggressiveness of P. capsici isolates on pumpkin and pepper.
Molecular studies. The PCR product of the ITS regions, amplified with primers 5 and 4, produced 840-bp fragments for P. capsici and 980-bp fragments for P. sojae. Seven out of fifteen enzymes tested digested the PCR products. The results did not show any obvious difference in bands among the isolates of P. capsici; whereas, differences in bands between P. capsici and P. sojae were clearly evident.
In the ISSR test, 11 of 20 primers [(AG)8T, (AG)8C, (GA)8T, (CA)8G, (AC)8T, (AG)8YT, (AC)8YC, (GACA)5, (GTC)7, (ACG)7, (CAT)7] (Table 2) generated clearly different band profiles. Each of the primers resulted in different band pattern. Modification of PCR amplification conditions did not improve the band patterns. The number of bands produced by amplified product of the genomic DNA ranged from 5 to14, with size ranging from 300-bp to 3000-bp. A total of 77 reproducible polymorphic bands were detected for all 11 primers. Isolates from different locations produced different band patterns.
Analysis of genetic variation of 24 isolates using 22 primers in the AFLP tests (Table 2) resulted in clear differences among the isolates. A total 147 reproducible polymorphic bands were detected, with the size ranging from 200-bp to 2000-bp in length. Each of the selected primers resulted in a different band pattern.
When the results of ISSR and AFLP tests were combined, the relationships depicted in the phenogram indicated that the isolates were separated into three distinct groups, based on the location where they had been collected. Group I included the isolates from Pekin and Manito in central Illinois, group II contained the isolates from Shawneetown in southern Illinois, and group III included the PP1 and PP2 isolate from Helongjiang province in China. There was no relationship between virulence or mating types of the isolates and grouping of the isolates in the phenogram.
Genetic analysis of the ITS regions couldn’t differentiate P. capsici from each other. But, the genetic markers in ISSR and AFLP tests were powerful tools in revealing genetic differences among isolates of P. capsici. Both ISSR and AFLP tests produced similar grouping of the isolates. Therefore, both ISSR and AFLP are reliable methods for differentiating isolates of P. capsici based on genetic differences. The results of ISSR and AFLP tests showed that the isolates from the same region were closely related in their genetic composition than from different regions. These findings agree with reports by Foster et al. (7) and Lamour & Hausbeck (24) indicated significant variation of P. capsici from different locations. The results may indicate that geographically separated sites have different pools of genetic diversity and that long distance dispersal of P. capsici may not be common.
This study was the first use of modified AFLP protocol developed by Alonso (1) to analysis genetic diversity among isolates of P. capsici. The results showed that this protocol can be used to differentiate genetic variation among P. capsici isolates.
The results of this study and reports by other investigators (7,24) showed that there are significant differences in virulence and genetics among isolates of P. capsici. Thus, in selecting plants for resistance to P. capsici, and developing strategies for management of this pathogen, large pools of P. capsici isolates from different locations should be considered.
Table 1. Source and mating types of Phytophthora capsici isolates used in this study.
Isolatez, Host, Field location,
Mating type, Year isolated
Pc2 Pumpkin, seedling Pekin, IL A1 2000
Pc7 Squash, stem Pekin, IL A1 2000
Pc16 Pumpkin, petiole Pekin, IL A2 2000
Pc19 Muskmelon, stem Shawneetown, IL A1 2000
Pc23 Cucumber, fruit Shawneetown, IL A1 2000
Pc25 Watermelon, fruit Shawneetown, IL A2 2000
Pc26 Cucumber, fruit Shawneetown, IL A2 2000
Pc27 Melon, fruit Shawneetown, IL A1 2000
Pc28A Pepper, stem Shawneetown, IL A2 2000
Pc29 Eggplant, fruit Shawneetown, IL A1 2000
Pc30 Nightshade, stem Shawneetown, IL A1 2000
Pc32 Watermelon, fruit Shawneetown, IL A1 2001
Pc35#4z Pumpkin, petiole Pekin, IL A2 2001
Pc35#6z Pumpkin, petiole Pekin, IL A2 2001
Pc35#7z Pumpkin, petiole Pekin, IL A2 2001
Pc35#9z Pumpkin, petiole Pekin, IL A2 2001
Pc35#10z Pumpkin, petiole Pekin, IL A2 2002
Pc42#1 Pumpkin, stem Manito, IL A1 2002
Pc43#2 Pumpkin, stem Manito, IL A2 2002
Pc44#4 Zucchini, petiole Shawneetown, IL A1 2002
Pc51#4 Zucchini, petiole Shawneetown, IL A1 2002
Pc57#6 Pepper, stem Shawneetown, IL A2 2002
PP1 Pumpkin, stem Harbin, Heilongjiang, China A1 2001
PP2 Pepper, stem Beian, Heilongjiang, China A1 2001
____________________________________________________
z Isolates marked with the same letter were collected from the same field, and
each of the other isolates was collected from a different field.
Table 2. Source and mating types of P. capsici isolates used for inoculations.
Isolates, Source, Mating type
Pc-15 Pumpkin petiole A2
Pc-20 Pumpkin seedling A1
Pc-24B Pumpkin fruit A1
Pc-35#4 Pumpkin petiole A2
Pc-34#7 Pumpkin petiole A2
Pc-38#15 Pumpkin petiole A1
Table 3. Primers used in inter-simple sequence repeats (ISSR) amplification and amplified fragment-length polymorphism (AFLP) assays.
ISSR test, AFLP test, Primer, Sequence, Primer, Sequence
1 (AG)8T 1 GACTGCGTACCAATTC CAT
2 (AG)8C 2 GACTGCGTACCAATTC CATA
3 (GA)8T 3 GACTGCGTACCAATTC CATC
4 (CA)8G 4 GACTGCGTACCAATTC CATG
5 (AC)8T 5 GACTGCGTACCAATTC CCG
6 (AG)8YT 6 GACTGCGTACCAATTC CCGA
7 (AC)8YC 7 GACTGCGTACCAATTC CCGG
8 (GACA)5 8 GACTGCGTACCAATTC CCGT
9 (GTC)7 9 GACTGCGTACCAATTC CGC
10 (ACG)7 10 GACTGCGTACCAATTC CGCA
11 (CAT)7 11 GACTGCGTACCAATTC CGCT
12 (AC)10 12 GACTGCGTACCAATTC CTC
13 (AT)10 13 GACTGCGTACCAATTC CTCA
14 (GT)10 14 GACTGCGTACCAATTC CTCC
15 (GC)10 15 GACTGCGTACCAATTC CTCG
16 (TC)10 16 GACTGCGTACCAATTC CTCT
17 (CCA)7 17 GACTGCGTACCAATTC GCG
18 (TTA)7 18 GACTGCGTACCAATTC GCGA
19 (TAG)7 19 GACTGCGTACCAATTC GCGC
20 (ATA)7 20 GACTGCGTACCAATTC AGC
21 GACTGCGTACCAATTC TGC
22 GACTGCGTACCAATTC GAT
Table 4. Susceptibility of 45 plant species to six P. capsiciv isolated from pumpkin. Plant, Plants infected (%), Re-isolation, PCR detection.
Family, Common name, Scientific name, Cultivar, 3 days, 10 days
Chenopodiaceae Beetw Beta vulgaris Ruby Queen 21.7 55.6 +x Negative
Spinach Spinacia olerace Old Dominion 41.7 83.9 + Positive
Swiss-chardw Beta vulgaris var. cicla Rhubarb 24.9 64.8 + Negative
Cruciferae Radish Raphanus sativus. French Breakfast 21.6 60.8 + Positive
Turnipw Brassica rapa Purple Top 27.7 53.9 + Positive
Cucurbitaceae Cantaloupe Cucumis melo Sweet Granite 80.5 100 + Positive
Cucumber Cucumis sativus Cayenne 75.9 100 + Positive
Gourd Cucurbita pepo Bird House 66.9 95.9 + Positive
Honeydew melon Cucurbita melo Honey Roch 80.7 100 + Positive
Melon Pisum melo Annanas 88.6 100 + Positive
Squash Cucurbita pepo Sebring F1 88.6 100 + Positive
Watermelon Citrullus lanatus SWT6703 80.7 100 + Positive
Zucchini Cucurbita pepo Dark Green 90.9 100 + Positive
Leguminosae Green beanw Phaseolus vulgaris Bush Blue Lake 30.8 52.6 + Positive
Lima beansw Phaseolus lunatus Ford Hook 242 31.6 63.8 + Positive
Snow Pea Pisum sativus Snow Flake 10.6 51.9 + Negative
Liliaceae Onion Allium cepa Red Wether Field 20.6 41.9 + Positive
Malvaceae Velvet leafw y Abutilon theophrasti 34.9 78.3 + Positive
Solanaceae Eggplant Solanum melongena Classic 35.6 75.8 + Positive
Nightshadew Solanum nigrum 45.7 92.9 + Positive
Pepper Capsicum annuum California wonder 51.4 100 + Positive
Tobacco Nicotiana tabacum Sacred 24.9 70.7 + Positive
Tomato Lycopersicon esculentum Popreco 45.8 85.7 + Positive
Umbelliferae Carrot Daucus carota Red Core Chantanay 30.6 85.9 + Positive
Amaranthaceae Pigweed y Amaranthus etroflexus 0 0 - Negative
Water hemp y Amaranthus rudis 0 0 - Negative
Chenopodiaceae Lambquaters y Chenopodium album 0 0 - Negative
Compositae Cocklebur depined y Xanthium strumarium 0 0 - Negative
Cruciferae Broccoli Brassica oleracea Nomad 0 0 - Negative
Cabbage Brassica oleracea Jersey Wakefield 0 0 - Negative
Cauliflower Brassica oleracea Snow Ball X 0 0 - Negative
Kale Brassica oleracea White Russian 0 0 - Negative
Kohlrabi Brassica oleracea Early White Vienna 0 0 - Negative
Mustard Brassica nigra Tatsoi 0 0 - Negative
Gramineae Corn Zea mays Wisconsin Black 0 0 - Negative
Wheat Triticum aestivum Clark 0 0 - Negative
Labiatae Basil Ocimum basilicum Thai 0 0 - Negative
Leguminosae Soybeanz Glycine max 0 0 - Negative
Liliaceae Chives Allium schoenoprasum Herb 0 0 - Negative
Poaceae Crab grass y Digitaria sanguinalis 0 0 - Negative
Sandbur y Cenchrus incertus 0 0 - Negative
Umbelliferae Celery Apium graveolens Giant Red 0 0 - Negative
Dill Anethum graveolens Long Island 0 0 - Negative
Parsley Petroselinum crispum Moss Curled 0 0 - Negative
Zygophyllaceae Puncture vine y Tribulus terrestris 0 0 - Negative
___________________________________________________
v Combined inocula of six isolates was used in inoculation.
w First report as a host of P. capsici.
x + = infected; - = uninfected.
y Weed species.
z Sixteen cultivars of soybean were tested.
Table 5. Analysis of variance for areas under disease progress curves(AUDPC)x on pumpkins inoculated with six isolates of Phytophthora capsici.
Source, df, y, Mean square, P>F
Experiment 1 21,371.6 0.071
Reps( Experiment) 3 6,828.6 0.531
Type × experiment 1 9,908.4 0.270
Cultivar × experiment 5 18,536.0 0.062
Type × cultivar x experiment 5 16,810.5 0.125
Isolate× experiment 5 10,435.9 0.271
Typez 1 206,668.7 <0.001
Type × isolate× experiment 5 3,313.7 0.746
Cultivar (type) 2 9,090.3 0.235
Isolate 5 39,537.8 <0.001
Type × isolate 5 36,113.9 <0.001
Isolate × cultivar( type) 10 25,372.0 <0.001
Isolate× cultivar × experiment 25 5,615.1 0.585
xAUDPC = n Σ i = 1 (Xi+1 + Xi) (ti+1 – ti)/2, where Xi = disease incidence at the ith observation, ti = days at the ith observation, and n = total number of observations.
__________________________________________________
y Degree of freedom
z Jack-o-lantern and processing pumpkins
Table 6. Areas under disease progress curves (AUDPC) on six pumpkin cultivars in an evaluation of virulence of six Phytophthora capsici from pumpkin.
Isolates, AUDPCy on different cultivars, Jack-o-lantern pumpkins, Processing pumpkins,
Gold rush, Gold medal, Pik-A-pie,
Dickinson, Hybrid-401, Hybrid-698
Pc-15 216.8az 216.8a 208.3bc 186.7a 168.3b 188.4a
Pc-20 213.2ab 206.7abc 216.6ab 156.8b 175.8ab 172.5b
Pc-24B 226.7a 208.3ab 226.7a 191.7a 180.0a 201.8a
Pc34#7 218.4a 201.6bcd 200.0bc 158.4b 172.5ab 166.7bc
Pc35#4 191.7c 190.0d 196.7c 143.3c 155.0c 155.8c
Pc38#15 200.0bc 195.0cd 201.8bc 152.5b 152.5c 163.3bc
LSD(P=0.05) 15.8 12.9 17.4 9.1 11.4 13.4
yAUDPC = n Σ i = 1 (Xi+1 + Xi) (ti+1 – ti)/2, where Xi = disease incidence at the ith observation, ti = days at the ith observation, and n = total number of observations.
z In each column, the value with a letter in common are not significantly different from each other according to Fisher’s protected LSD(P=0.05)
Table 7. Area under disease progress curve (AUDPC) on six host plants inoculated with Phytophthora capsici isolates from different hosts and locations.
Isolatew, AUDPCx, Mean, AUDPC, LSD (P=0.05), Pumpkin, Squash, Watermelon, Pepper,
Eggplant, Tomato
Pc2 205.7 235.5 185.1 154.2 132.2 138.7 175.2 jy 26.7
Pc7 238.0 194.6 243.6 177.7 164.7 173.5 198.5 b-e 23.2
Pc16 167.1 218.1 212.2 167.1 144.7 168.5 179.6 h-j 28.6
Pc19 182.8 222.7 203.2 187.5 166.3 197.1 183.3 f-j 19.9
Pc23 188.7 189.8 191.5 179.5 148.2 163.5 176.9 ij 12.6
Pc25 270.5 218.1 276.1 191.6 188.7 195.5 223.4 a 32.4
Pc26 183.5 197.2 194.4 153.6 143.9 172.5 174.2 j 18.7
Pc27 242.7 221.5 211.6 187.5 167.4 169.8 200.1 b-e 23.4
Pc28A 243.6 216.7 218.5 192.2 176.8 189.5 205.8 bc 15.9
Pc29 198.7 191.6 196.1 170.6 151.5 179.5 181.3 g-j 17.2
Pc30 194.5 186.8 197.7 163.8 166.7 178.6 181.5 g-j 14.6
Pc32 192.2 195.7 207.1 184.8 178.2 199.9 192.9 b-h 11.4
Pc35#4 199.9 189.6 208.3 186.5 168.1 176.3 188.1 d-j 20.3
Pc35#6 205.5 190.2 211.7 172.7 178.6 180.6 189.8 d-j 16.7
Pc35#7 209.8 207.2 222.7 202.9 185.9 173.5 200.2 b-e 21.1
Pc35#9 211.9 198.8 233.5 210.5 193.8 203.8 208.7 ab 10.5
Pc35#10 197.7 205.3 236.4 187.5 177.5 185.5 198.3 b-f 19.6
Pc42#1 253.3 197.7 227.5 171.7 154.3 187.2 192.6 c-h 17.9
Pc43#2 234.7 198.8 191.2 160.6 158.1 164.5 184.6 e-j 23.8
Pc44#4 201.4 183.5 191.6 176.5 165.1 181.5 183.2 f-j 21.4
Pc51#4 237.3 219.7 203.7 188.3 175.4 166.4 198.5 b-e 18.7
Pc57#6 210.2 182.6 191.6 189.1 187.5 179.3 190.1 c-j 11.2
PP1 216.2 219.7 204.4 186.4 176.7 179.3 197.1 b-g 21.6
PP2 229.7 252.9 216.3 192.1 135.9 192.6 203.2 b-d 18.6
Mean 213.2az 205.6a 211.5a 180.6b 166.1c 179.0b
LSD (P=0.05) 34.7 32.1 31.4 28.7 26.9 24.6 15.9
w Isolates marked with the same letter were collected from the same field, and each of the other isolates was collected from a separate field.
___________________________________________________
x AUDPC = n Σ i = 1 (Xi + 1 + Xi)(ti + 1 – ti)/2, where X i = Percent of plant death at the ith observation, ti = days at the ith observation, and n = total number of observations. AUDPC values represent mean values of two experiments.
y Values in the column with a letter in common are not significantly different from each other according to Fischer’s protected LSD (P = 0.05).
z Values in the row with a letter in common are not significantly different from each other according to Fischer’s protected LSD (P = 0.05).
Educational & Outreach Activities
Participation Summary:
The results of this research were presented to growers, extension personnel, agribusiness personnel, IR-4 staff, and scientists in regional, statewide, national, and international meetings. The following are some of the meetings at which results were presented:
1. Specialty Crop Growers Meeting (Illinois) – 2003.
2. Arthur Vegetable Growers Meeting (Illinois) – 2003.
3. IPM specialists (Illinois) – 2003.
4. Kankakee Vegetable Growers School (Illinois) – 2003.
5. Summer Pumpkin Day (Illinois) – 2003.
6. Adams Country Vegetable and Fruit Growers School (Illinois) – 2003.
7. Summer Horticulture Day (Illinois) – 2003.
8. Southern Illinois Vegetable School (Illinois) – 2003.
9. Annual Meeting of the American Phytopathological Society, Charlotte, NC – 2003.
10. Illinois-Indiana Vegetable Growers Meeting – 2004.
11. Illinois-Wisconsin Vegetable-Fruit Growers Meeting – 2004.
12. Illinois-Iowa Vegetable-Fruit Growers Meeting – 2004.
13. Southern Vegetable Growers Meeting (Illinois) – 2004.
14. International Pepper Conference in Florida – 2004.
15. Food Processors Conference in London Canada – 2005.
16. Pumpkin IPM for pumpkin growers, agribusiness personnel, and extension/research specialist of Illinois, Indiana, Iowa, and Missouri – 2005.
17. Pepper and Tomato Growers Meeting in St. Catherine, Canada – 2005.
18. Vine Crop Growers Meeting in St. Catherine, Canada – 2005.
19. IR-4 Phytophthora capsici Conference in Little Rock, Arkansas – 2005.
In additions to the above-mentioned presentations, several extension specialists in New York, Texas, and Canada obtained the results of this research for presentation to the growers in respected areas.
The following are published papers that include results of this research:
1. Babadoost, M. 2004. Phytophthora blight of cucurbits: how to manage it? The Specialty Growers News June 2004:15.
2. Babadoost, M. 2004. Phytophthora blight: A serious threat to cucurbit industries. http://www.apsnet.org/online/feature/cucurbit/.
3. Babadoost, M., and S.Z. Islam. 2004. Methods for managing Phytophthora blight (Phytophthora capsici) of pepper. The 17th International Pepper Conference, November 14-16, Naples, Florida. Page 1 in the Program and Abstracts, 33pp.
4. Tian, D., and M. Babadoost. 2003. Determining the host range of Phytophthora capsici in Illinois. Phytopathology 93:S83.
5. Tian, D., and M. Babadoost. 2003. Genetic variation among isolates of Phytophthora capsici from Illinois. Phytopathology 93:S84.
6. Babadoost, M. 2004. Phytophthora blight of cucurbits. 3 pp (Fact Sheet).
7. Tian, D., and M. Babadoost. 2004. Host range of Phytophthora capsici from pumpkin and pathogenicity of the isolates. Plant Dis. 88:485-489.
Project Outcomes
In this study, 45 species of crop and weed plants were screened for their susceptibility to P. capsici. Twenty-two crop species (beet, carrot, eggplant, green bean, lima bean, radish, snow pea, spinach, Swiss-chard, tomato, turnip, onion, pepper and a long list of vine vegetables including pumpkin, cantaloupe, cucumber, gourd, honeydew melon, muskmelon, squash, watermelon and zucchini) succumbed to the disease, 14 did not. The results of this research will help growers to establish effective cropping rotations for managing P. capsici in vegetable and weed control, thus establishing sustainable vegetable, particularly cucurbit, production.
Economic Analysis
Illinois, with approximately 22,000 acres of pumpkin, is the leading state in pumpkin production. Also about 10,000 acres of cantaloupe, cucumbers, eggplants, pepper, tomatoes, and watermelon are grown in Illinois. These vegetables are widely grown in the North Central Region and nationwide. About 90% of commercial processing pumpkins produced in the United State (US) are grown in Illinois. Michigan is the home of a major cucumber pickling industry. Phytophthora blight, caused by P. capsici, has become one of the most serious threats to production of cucurbits, eggplants, pepper, and tomatoes. The disease causes up to 100% yield losses in commercial fields. Value of crop/product losses in pumpkin and pepper industry in Illinois alone, can exceed $10,000,000 per year. The results of this research are expected reduce crops losses in vegetable fields to P. capsici, which will allow the vegetable industry to be a significant component of the agricultural economy in Illinois, the North Central Region, and nationwide.
Farmer Adoption
The results of this research on host range of P. capsici have been widely used by growers in Illinois and likely in other states. Farmer adoption minimizes crop losses to this pathogen in appropriate cropping systems. Based on the findings of this research on pathogenic genotypic diversity of P. capsici, breeders intend to use a larger pool of isolates of this pathogen in their breeding programs, which will benefit vegetable growers.
Areas needing additional study
The following areas of research study are suggested:
1. Develop reliable methods for extraction of Phytophthora capsici from soil.
2. Develop reliable methods for evaluating viable teliospores of Phytophthora capsici.
3. Determine survival of Phytophthora capsici in soil.
4. Develop resistant cultivars of cucurbits and peppers against Phytophthora capsici.
5. Develop effective IPM strategies against Phytophthora capsici in commercial fields.