Taxonomical Identification of Clarireedia Species Causing Dollar Spot Disease of Turfgrass in Georgia

Abstract

Dollar spot caused by the fungal pathogen Clarireedia spp, is an important disease of all warm and cool season grasses that are grown in the US, including the state of Georgia. Disease symptoms similar to those of dollar spot i.e., white to straw-colored circular lesions on leaf blades and large irregular sunken patches on turf swards, were regularly observed with approximately 50% incidence during spring and fall seasons spanning from early March to late May as well as September to November in the turfgrass fields located at University of Georgia-Griffin Campus, Griffin, GA (Latitude: 33.26, Longitude: -87.27). To isolate and identify the pathogens, symptomatic plant samples were collected in September of 2019 from bermudagrass (Cynodon dactylon), creeping bentgrass (Agrostis palustris syn. A. stolonifera), zoysiagrass (Zoysia japonica), and seashore paspalum (Paspalum vaginatum). Center portions of the diseased leaves were cut into small pieces (1-2 cm), surface disinfected (10% bleach for 2 min, 80% ethanol for 2 min, and rinse with sterile water for three times), and dried within a sterile hood for about 5 mins. Disinfected leaf segments were plated on potato dextrose agar (PDA) and incubated at room temperature. The development of white fluffy mycelium was visible for all isolates plated after 24-h of incubation. DNA was isolated from all four isolates using cetyltrimethylammonium bromide (CTAB) method (Doyle and Doyle 1987) and the internal transcribed spacer (ITS) regions of the ribosomal DNA was amplified using ITS4 and ITS5 primers (White et al. 1990). Based on the sequence comparison in the NCBI database, the isolate collected from creeping bentgrass was identified as Clarireedia jacksonii with 100% sequence homology to the reference MF964320; while the three isolates collected from bermudagrass, zoysiagrass, and seashore paspalum were all identified as Clarireedia monteithiana with 99.60%, 100%, and 99.60% sequence homology to the reference KF545806, respectively. The ITS sequences of the isolates collected from creeping bentgrass, bermudagrass, zoysiagrass, and seashore paspalum were deposited in GenBank with accession number MT497874, MT497855, MT497856, and MT497854, respectively. Furthermore, based on sequence alignment with ClustalW, the fungal isolate obtained from creeping bentgrass had all the 28 single nucleotide polymorphism (SNP) combination specific to C. jacksonii, and three isolates collected from bermudagrass, zoysiagrass, and seashore paspalum had all SNP combination specific to C. monteithiana (Salgado-Salazar et al. 2018). Our results support the host specialization described by Salgado-Salazar et al. (2018) in natural infections, where Clarireedia jacksonii was identified on cool-season turfgrass while C. monteithiane was uniquely found on warm-season species of turfgrass. Pathogenicity tests were conducted on bermudagrass ‘Princess’, creeping bentgrass ‘Penn A1/A4’, zoysiagrass ‘Zorro’, and seashore paspalum ‘Seastar’ with three replications for each treatment. The plant materials were prepared in Kord Presto sheet pots (10 cm × 10 cm) filled with Sungro professional growing mix soil (Sun Gro Horticulture Distribution Inc.). A single mycelial plug from a pure culture of the pathogens was transferred into a sterile 125-ml Erlenmeyer flasks containing 100 ml potato dextrose broth and shook at a speed of 150 rpm at room temperature. The 7-days old mycelium suspension of each isolate was used to inoculate the plant materials (all four turfgrass species) using a Research Track Sprayer (Devries Manufacturing Inc.) and then the inoculated plants were placed in a dew chamber with high humidity for 48 h. Enough spacing between the treatments was provided inside the due chamber to prevent accidental contaminations. Controls plants were inoculated with potato dextrose broth using a Research Track Sprayer. Plants were then transferred to a greenhouse bench. Typical symptoms of dollar spot (white/tan lesions and sunken patches) were visible 5 days after inoculation while the control plants had no disease development. Furthermore, all isolates of the dollar spot pathogen used in the inoculation procedures were able to cross-infect all four turfgrass species. The pathogens, C. jacksonii and C. monteithiana were re-isolated from the artificially inoculated plants. To our knowledge, this is the first report on the taxonomical identification of C. jacksonii and C. monteithiana pathogens causing dollar spot disease on turfgrass in Georgia, the first report on taxonomical identification of C. monteithiana on zoysiagrass in the US, and the first report of cross-infect potential of Clarireedia species in the greenhouse under artificial inoculation. This information will be useful in effective identification and management of dollar spot disease of turfgrass.

Full text

Disease Notes
Diseases Caused by Fungi and Fungus-Like Organisms
Taxonomical Identification of Clarireedia Species Causing Dollar Spot
Disease of Turfgrass in Georgia
S. Sapkota,
1,2
A. D. Martinez-Espinoza,
1
E. Ali,
3
C. B. Vermeer,
1
and
B. A. Bahri
1,2,†
1
Department of Plant Pathology, University of Georgia, Griffin, GA 30223
2
Institute of Plant Breeding, Genetics, and Genomics, University of
Georgia, Griffin, GA 30223
3
Department of Plant Pathology, University of Georgia, Tifton, GA 31794
Funding: This study was supported by Hatch Act and State of Georgia
funding. Plant Dis. 104:30 3, 2020; published online as https://doi.org/
10.1094/PDIS-03-20-0603-PDN. Accepted for publication 6 June 2020.
Dollar spot caused by Clarireedia spp. is an important disease of all
warm and cool season grasses in the USA, including Georgia. Symptoms
(i.e., white to straw-colored circular lesions on leaf blades and large
irregular sunken patches on turf swards) were regularly observed with
;50% incidence from early March to late May as well as September to
November in the turfgrass fields at University of Georgia–Griffin Campus
(lat., 33.26; long., –87.27). Symptomatic plant samples were collected in
September 2019 from bermudagrass (BG) (Cynodon dactylon), creeping
bentgrass (CB) (Agrostis palustris syn. A. stolonifera), zoysiagrass (ZG)
(Zoysia japonica), and seashore paspalum (SP) (Paspalum vaginatum).
Center portions of diseased leaves were cut into 1- to 2-cm pieces, surface
disinfected (10% bleach for 2 min, 80% ethanol for 2 min, and rinsed with
sterile water three times), and dried in a sterile hood for ;5 min.
Disinfected leaf segments were plated on PDA and incubated at room
temperature (RT). White fluffy mycelium was visible for all isolates after
24 h of incubation. DNA was isolated from all four isolates using the
CTAB method (Doyle and Doyle 1987), and the rDNA ITS region was
amplified using ITS4/ITS5 primers (White et al. 1990). Based on sequence
comparison in the NCBI database, the isolate from CB was identified as
Clarireedia jacksonii with 100% sequence homology to MF964320,
whereas the isolates from BG, ZG, and SP were identified as Clarireedia
monteithiana with 99.60, 100, and 99.60% sequence homology to
KF545806, respectively. The ITS sequences of the isolates collected from
CB, BG, ZG, and SP were deposited in GenBank: MT497874, MT497855,
MT497856, and MT497854, respectively. Furthermore, based on sequence
alignment with ClustalW, the isolate from CB had the combination of all 28
single nucleotide polymorphisms (SNPs) specific to C. jacksonii, and
isolates collected from BG, ZG, and SP all had the SNP combination
specific to C. monteithiana (Salgado-Salazar et al. 2018). Our results
support the host specialization described by Salgado-Salazar et al. (2018) in
natural infections: C. jacksonii was identified on cool-season turfgrass,
whereas C. monteithiana was uniquely found on warm-season species.
Pathogenicity tests were conducted on BG ‘Princess’,CB‘Penn A1/A4’,
ZG ‘Zorro’, and SP ‘Seastar’with three replications for each treatment.
Plant materials were prepared in Kord Presto sheet pots (10 × 10 cm) filled
with Sun Gro professional growing mix. A single mycelial plug from a pure
culture of the pathogens was transferred into a sterile 125-ml Erlenmeyer
flask containing 100 ml of potato dextrose broth and shaken at 150 rpm at
RT. The 7-day-old mycelium suspension of each isolate was used to
inoculate all four turfgrass species using a research track sprayer (Devries
Manufacturing), and then the inoculated plants were placed in a dew
chamber with high humidity for 48 h. Enough spacing between the
treatments was provided inside the dew chamber to prevent accidental
contamination. Controls plants were inoculated with potato dextrose broth
using a research track sprayer. Plants were transferred to a greenhouse
bench. Typical symptoms of dollar spot (white/tan lesions and sunken
patches) were visible 5 days after inoculation; the control plants had no
disease development. All isolates of the dollar spot pathogen used were
able to cross-infect all four turfgrass species. C. jacksonii and C.
monteithiana were reisolated from the artificially inoculated plants. This
is the first report on the taxonomical identification of C. jacksonii and C.
monteithiana pathogens causing dollar spot on turfgrass in Georgia, the
first report on taxonomical identification of C. monteithiana on ZG in the
USA, and the first report of cross-infection potential of Clarireedia species
in the greenhouse under artificial inoculation. This information will be
useful in effective identification and management of dollar spot disease of
turfgrass.
References:
Doyle, J. J., and Doyle, J. L. 1987. Phytochem. Bull. 19:11.
Salgado-Salazar, C., et al. 2018. Fungal Biol. 122:761.
White, T. J., et al. 1990. Page 315 in: PCR Protocols: A Guide to Methods and
Applications. Academic Press, San Diego, CA.
The author(s) declare no conflict of interest.
e-Xtra
Keywords: internal transcribed spacer (ITS), single nucleotide
polymorphism (SNP), pathogenicity, cross-infection
†
Indicates the corresponding author.
E-mail: bbahri@uga.edu (B.A.B.)
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