Variations in virulence and hyphal growth of four Raffaelea quercivora isolates within Quercus crispula

Abstract

Mass mortality of fagaceous trees caused by Japanese oak wilt has occurred widely in Japan. Although virulence of the causal fungus, Raffaelea quercivora, appeared to differ among isolates, its relation to the fungal growth within trees was unknown. To clarify the differences in fungal virulence against susceptible Quercus crispula, we examined fungal growth of four R. quercivora isolates within trees and the resulting virulence. In our study, the isolates were multiple-inoculated in seedlings and single-inoculated in twigs of mature trees. In the multiple-inoculation test, mortality rates were examined by the observation of external symptoms. In the single-inoculation test, water conductance and hyphal growth within the trees were examined by applying aqueous dyes and fluorescence microscopy, respectively. Mortality rates, the proportion of the cross-sectional area comprising non-conductive sapwood and horizontal hyphal growth differed significantly among the isolates. Univariate logistic regression analyses showed that both the proportion of non-conductive sapwood and hyphal growth were significantly positively related to mortality rates. For three isolates, hyphal growth was significantly positively correlated with the proportion of non-conductive sapwood. These results suggested that the virulence against Q. crispula varies among R. quercivora isolates and that the extent of fungal colonization of the tree determines fungal virulence.

Variation in virulence and its relation to hyphal growth and enzyme activity of Raffaeleaquercivora, a Japanese oak wilt pathogen

“…quercivora collected from 13 host species growing in 14 prefectures in areas affected by JOW in Japan (Figure 1, Table S1). Except for isolates whose virulence had been evaluated in previous studies (Kusumoto et al, 2015; Torii et al, 2016) and the type strain of R . quercivora , all isolates were identified as R .…”

“…10 mm and inserted a fungus‐colonized toothpick (5 mm length, 1.5 mm diameter) into the hole. We chose 9 of the 40 isolates that originated from different geographical regions and displayed variation in virulence in prior studies (Kusumoto et al, 2015; Torii et al, 2016). To minimize variation in host defence responses between individual trees, we used 10 twigs of the same tree for all 9 isolates and the control.…”

“…We used 40 monospore isolates of R. quercivora collected from 13 host species growing in 14 prefectures in areas affected by JOW in Japan (Figure 1, Table S1). Except for isolates whose virulence had been evaluated in previous studies (Kusumoto et al, 2015;Torii et al, 2016) and the type strain of R. quercivora, all isolates were identified as R. quercivora on the basis of the partial nucleotide sequences of the 28S large subunit rDNA (D1/D2) following the method of Torii et al, (2016). All newly obtained sequences (499 bp) matched completely the fungus isolates with DDBJ accession numbers LC069311 and LC069313.…”

Comparisons of hyphal growth of Raffaelea quercivora among four Japanese Fagaceae species

“…In both tests, inocula were prepared by culturing wooden sticks with or without the fungus as described by Torii et al. () except for using the fungal isolate RA2117, which was preserved in the Laboratory of Forest Pathology and Mycology, Mie University. In Experiment 1, eight seedlings of each species were inoculated on 9 July 2012.…”

“…() detected most hyphae in non‐conductive sapwood under both artificial and natural conditions. Moreover, Torii, Matsuda, and Ito (), Torii, Ito, Nagao, Matsuda, and Ito () suggested that the extent of mycelial colonization was proportional to the amount of non‐conductive sapwood.…”