Development of an Improved Isolation Approach and Simple Sequence Repeat Markers To Characterize Phytophthora capsici Populations in Irrigation Ponds in Southern Georgia

Abstract

Phytophthora capsici, the causal agent of Phytophthora blight, is a major concern in vegetable production in Georgia and many other states in the United States. Contamination of irrigation water sources by P. capsici may be an important source of inoculum for the pathogen. A simple method was developed in this study to improve the efficiency of recovering P. capsici from fruits used as baits in irrigation ponds. In contrast to direct isolation on agar plates, infected fruit tissues were used to inoculate stems of pepper seedlings, and the infected pepper stems were used for isolation on agar plates. With isolation through inoculation of pepper stems, the frequency of recovering P. capsici from infected eggplant and pear fruits increased from 13.9% to 77.7% and 8.1% to 53.5%, respectively, compared with direct isolation on agar plates. P. capsici was isolated from seven out of nine irrigation ponds evaluated, with most of the ponds containing both A1 and A2 mating types and a 4:5 ratio of A1 to A2 when isolates from all ponds were calculated. All P. capsici isolates were pathogenic on squash plants, and only a small proportion (8.2%) of the isolates were resistant or intermediately sensitive to mefenoxam. Simple sequence repeats (SSRs) were identified through bioinformatics mining of 55,848 publicly available expressed sequence tags of P. capsici in dbEST GenBank. Thirty-one pairs of SSR primers were designed, and SSR analysis indicated that the 61 P. capsici isolates from irrigation ponds were genetically distinct. Cluster analysis separated the isolates into five genetic clusters with no more than two genetic groups in one pond, indicating relatively low P. capsici genetic diversity in each pond. The isolation method and SSR markers developed for P. capsici in this study could contribute to a more comprehensive understanding of the genetic diversity of this important pathogen.

Genetic diversity of Phytophthora capsici (Pythiaceae) isolates in Anhui Province of China based on ISSR-PCR markers

“…Using molecular methods to investigate the genetic structure of P. capsici still drew a blank until the late 1980s and early 1990s (Förster et al, 1989;Oudemans and Coffey, 1991;Mchau and Coffey, 1995). Recently, molecular genetic markers, such as random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP), have been broadly used to learn the genetic structure of P. capsici (Lamour and Hausbeck, 2001;Silvar et al, 2006;Wang et al, 2009). Among the molecular markers, inter-simple sequence repeat (ISSR) is the easily usable and reliable one for investigating genetic diversity in plants, animals and fungi, and some studies have also shown that ISSR could provide high genomic polymorphism compared to restriction fragment length polymorphism, SSR and RAPD (Blair et al, 1999;Gilbert et al, 1999;Wang et al, 2009).…”

Soil amendments with Brassica cover crops for management of Phytophthora blight on squash

“…Efforts have been directed at developing disease‐resistant crop varieties; however, limited success has been achieved in developing resistant varieties, and no commercial cultivars of cucurbits resistant to the disease are available. While fungicides containing active ingredient mefenoxam provide some levels of control of the disease, mefenoxam‐resistant strains of P. capsici , which challenge the usefulness of this compound, have developed 5–8. Development of alternative or complementary methods for more efficient management of the disease is highly desirable.…”

Sensitivity of Phytophthora capsici on Vegetable Crops in Georgia to Mandipropamid, Dimethomorph, and Cyazofamid

“…A 7-mmdiameter plug removed from the edge of single colonies was placed on V8 agar and incubated at 25°C in the dark for 4 days. Pathogenicity of the isolates was determined by inoculation of squash plants with zoospores as described previously (34). Longterm storage of isolates was completed by putting a 7-mm mycelial plug of purified single-zoospore isolates in a 2-ml crj'ogenic vial containing one sterilized hemp seed and 1 ml of SDW, and the vials were incubated at 25°C for 2 weeks and stored at 15°C (7).…”

“…Singlezoospore isolates were grown on V8 agar in the dark for 7 days. DNA was extracted as described earlier (34) and polymerase chain reaction (PCR) amplification was performed using P. capsici-sptcific primers (PC-1, 5'-GTCTTGTACCCTATCATGGCG-3' and PC-2, 5'-CGCCACAGCAGGAAAAGCATT-3') to verify the identity of the isolates (37). PCR was performed in 20 pi of reaction mixture containing 20 ng of DNA, 200 pM each dNTP, 0.5 pM each primer, 0.5 U of Tag DNA polymerase, and lx PCR reaction buffer.…”