First Reports of Diaporthe kongii, D. masirevicii, and D. ueckerae Associated with Stem and Peg Dieback on Peanut in Australia

Abstract

In July 2016, peanuts (Arachis hypogaea L. cvs. Holt, Kairi) with symptoms of stem and peg dieback were collected from Kingaroy, Childers, and Bundaberg regions in Queensland, Australia. Pycnidia of Diaporthe spp. were visible in brown lesions on stems and on live and shriveled pegs. Affected tissues were surface sterilized in 1% v/v NaOCl for 3 min, rinsed with sterile distilled water, incubated on water agar with 100 µg/ml streptomycin sulfate at 23 to 25°C under ambient light. After 1 to 14 days, conidia were streaked onto potato dextrose agar with 100 µg/ml streptomycin sulfate (PDAS) and incubated as above. After 24 h, single germinating conidia were transferred to PDAS. Mycelial DNA was extracted from 80 single conidial isolates using the Bioline Isolate II Genomic DNA extraction kit. Primers for the internal transcribed spacer region (ITS) and partial region of the translation elongation factor 1-alpha (TEF) were as described in Thompson et al. (2015). Both ITS and TEF loci were amplified with Biolabs Hot Start Taq 2x Master Mix Enzyme. PCR products were purified using Bioline Sure Clean Plus and sequenced by Macrogen Incorporated (Seoul, Korea) with the amplification primers. Sequences were assembled with Geneious v.9.1. Species morphological descriptions are in Thompson et al. 2011, 2015, and Gao et al. 2016. Using BLAST sequence analysis and culture morphology, species were identified as D. kongii (four isolates), D. masirevicii (42), D. ueckerae (syn. D. miriciae) (27), or Diaporthe sp. (seven) (Gao et al. 2016; Thompson et al. 2011, 2015). A representative isolate of D. kongii (BRIP 64964d), D. masirevicii (BRIP 64786a), and D. ueckerae (BRIP 64785a) was deposited into the Queensland Plant Pathology Herbarium (Brisbane, Australia) and was used for pathogenicity testing. To verify pathogenicity, early podfill peanut plants (cv. Taabinga) grown at 21 to 25°C day, 14 to 18°C night, were stem slit inoculated (Thompson et al. 2011), using three plants per isolate. D. gulyae (BRIP 63295), a species pathogenic on sunflower and common in eastern Australia, was a positive control with negative controls mock inoculated with water agar. At the full seed to early maturity growth stage, lesions were rated on a 1 to 5 severity scale (SR) (Thompson et al. 2011). Inoculated stems were harvested, surface sterilized, and incubated as above. Isolates identified as D. kongii, D. masirevicii, and D. ueckerae were reisolated from pycnidia in lesions located 2 cm of the point of inoculation. In all cases, the reisolated culture morphology matched the inoculating culture. Negative controls were disease free. D. masirevicii and D. ueckerae were pathogenic on peanuts (cv. Taabinga) with moderate virulence (SR 3), D. kongii had low virulence (SR 2), and D. gulyae was pathogenic with high virulence (SR 4), causing significant stem dieback. Few Diaporthe (syn. Phomopsis) species have been recorded from peanuts. In India, Sharma (1974) isolated Phomopsis sp. from peanut leaf spots. Sanogo and Etarock (2009) reported P. longicolla causing peanut stem blight in New Mexico. To our knowledge, these are the first reports of D. kongii, D. masirevicii, and D. ueckerae on peanuts and are of significance as stem and peg infections potentially limit yield. Although not yet recorded on peanuts, D. gulyae is potentially a serious pathogen of peanuts.