Pathogenicity and molecular detection of nectriaceous fungi associated with black root rot of avocado

Black root rot of avocado associated with soilborne nectriaceous fungi is an aggressive disease of nursery trees and young orchards transplants, causing tree stunting, wilt, severe root necrosis, rapid decline and death within a year after planting. This study aimed to identify the fungal genera associated with the disease, determine the causal agents of black root rot, and develop a rapid molecular test for detection of key pathogens in avocado roots. A disease survey in all Australian growing regions collected 153 nectriaceous fungal isolates from roots of 91 symptomatic and healthy avocado trees and other hosts including peanut, papaya, blueberry, custard apple and grapevine. The fungal isolates were identified with phylogenetic analyses of ITS, β-tubulin and Histone H3 sequenced genes. Six genera were found associated with black root rot; Calonectria, Cylindrocladiella, Dactylonectria, Gliocladiopsis, Ilyonectria and Mariannaea. Glasshouse pathogenicity tests on 3–6 month-old avocado cv. Reed seedlings determined Calonectria ilicicola to be an aggressive pathogen, causing stunting and death within 5 weeks of inoculation. C. ilicicola isolated from peanut, papaya and custard apple also caused black root rot in avocado, demonstrating a broad host range. Calonectria sp. from blueberry and Dactylonectria macrodidyma, D. novozelandica, D. pauciseptata and D. anthuriicola from avocado caused significant root rot but not stunting within 5–9 weeks. Ilyonectria sp. from grapevine, and Cylindrocladiella pseudoinfestans, Gliocladiopsis peggii and Ilyonectria sp. isolates from avocado were determined to be non-pathogenic. Three loopmediated isothermal amplification (LAMP) assays were developed for the detection of C. ilicicola, D. macrodidyma and the Dactylonectria genus. The assays were sensitive and specific at DNA concentrations of 1 pg/µl, 0.01 ng/µl and 0.1ng/µl for C. ilicicola, D. macrodidyma, and Dactylonectria spp. respectively. Detection in avocado roots averaged from 12–26 min for C. ilicicola and D. macrodidyma and 14–30 min for Dactylonectria spp