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First Report of Diplodia sapinea and Diplodia scrobiculata Causing an Outbreak of Tip Blight on Slash Pine in Florida

Paez, C. A., Smith, J. A.
Plant disease 2018 v.102 no.8 pp. 1657
DNA, DNA primers, Diplodia pinea, Pinus elliottii, agar, air drying, autumn, blight, branches, cambium, culture media, decline, dieback, experimental design, financial economics, fungi, greenhouses, internal transcribed spacers, mortality, mycelium, pathogens, plastics, sapwood, seedlings, shoot tips, sodium hypochlorite, trees, virulence, wilting, Florida, Michigan
In the fall of 2012, extensive dieback and mortality were observed on slash pine stands along a highway in Orange County, FL (28°32′11.3″N, 81°33′30.1″W). Branches from symptomatic trees were collected and transported in a cooler to the laboratory. Small sapwood pieces from the margins of lesions from the symptomatic branches with resinous cankers on the stem were excised, surface sterilized in 2.5% sodium hypochlorite solution, and plated out on acidified potato dextrose agar (APDA; Difco Laboratories, Detroit, MI). Based on sequencing of the internal transcribed spacer (ITS-rDNA) using the primers ITS1 and ITS4 (White et al. 1990), Diplodia sapinea (GenBank accession no. MF111092 and NRRL no. 66756) and D. scrobiculata (GenBank MF111091 and NRRL 66757) were consistently isolated from symptomatic tissues. The recovery of these pathogens was possible in 56% of sampled trees. Diplodia tip blight is caused by the fungal pathogens D. sapinea and D. scrobiculata, which can also cause tree decline and cankers on stems (Paoletti et al. 2001). To confirm the pathogen, Koch’s postulates were conducted on 3-year-old seedlings of slash pine. Seedlings were wounded using a 6-mm sterile cork borer at ∼25 cm below the terminal shoot tip. Inoculation of the wounded stem was carried out with 6-mm agar plugs from the leading edge of an actively growing mycelial colony, facing the cambial tissue of the stem, and wrapped with Parafilm M (Pechiney Plastic). The experimental design was a randomized complete block design with three replicates and two fungal inoculation treatments (D. sapinea and D. scrobiculata), plus one noncolonized agar plug. The experiment was repeated twice in July 2015 and July 2016. Each time, the experimental unit was a single tree inoculated once in each replicate. The trees were maintained in the greenhouse and watered every 2 days. Parafilm was not removed until the end of this study. Lesion length and external symptoms were evaluated 11 weeks post inoculation. Pathogen reisolation was accomplished in one of each treatment by removal of sapwood (1 cm) from the margin between the live and dead tissue of the lesion with one replicate from each fungus, which was surface-sterilized in 2.5% sodium hypochlorite, followed by air-drying for 1 min before being plated on APDA. DNA was extracted and ITS regions were sequenced. D. sapinea produced significantly larger lesions than D. scrobiculata; average lesion length was 5.3 cm (4.9 to 7 cm range) and 2.4 cm (2 to 3 cm range), respectively. Wilting of new shoots and resinosis on the lower part of the stems were observed when inoculated with D. sapinea. Fungal treatments were significantly different from the controls (P < 0.05). The negative controls did not develop lesions or symptoms and the pathogen was not found after reisolation. D. sapinea is considered more virulent than D. scrobiculata (Bihon et al. 2011). This was also observed in our pathogenicity experiment, where D. sapinea was significantly more virulent than D. scrobiculata; however, either of these species can cause Diplodia tip blight (Luchi et al. 2005). This finding is significant because these pathogens have not been reported to cause outbreaks previously in the southeastern U.S. (whereas outbreaks on nonnative pines are common in the central and northern U.S.), and slash pine provides numerous ecological and economic benefits in the region (Schultz 1997). This disease could pose a new, serious threat to the resource.