Many Calonectria species are causal agents of diseases on several forestry, agricultural and horticultural crops. Calonectria leaf blight is one of the most important diseases associated with Eucalyptus plantations and nurseries in Asia and South America. Recently, symptoms of leaf rot and leaf blight caused by Calonectria species were observed in a one-year-old Eucalyptus experimental plantation in GuangXi Province, southern China. To better understand the species diversity, mating strategy and pathogenicity of Calonectria species isolated from diseased tissues and soils, diseased leaves and soils under the trees from ten Eucalyptus urophylla hybrid genotypes were collected. Three hundred and sixty-eight Calonectria isolates were obtained from diseased Eucalyptus leaves and soils under these trees, and 245 representative isolates were selected based on the sampling substrates and Eucalyptus genotypes and identified by DNA sequence analyses based on the translation elongation factor 1-alpha (tef1), β-tubulin (tub2), calmodulin (cmdA) and histone H3 (his3) gene regions, as well as a combination of morphological characteristics. These isolates were identified as Calonectria hongkongensis (50.2%), C. pseudoreteaudii (47.4%), C. aconidialis (1.6%), C. reteaudii (0.4%) and C. auriculiformis (0.4%). This is the first report of C. reteaudii and C. auriculiformis occurrence in China. Calonectria pseudoreteaudii was isolated from both Eucalyptus diseased leaves and soils; the other four species were only obtained from soils. MAT1-1-1 and MAT1-2-1 gene amplification and mating type assignment results showed that C. pseudoreteaudii is heterothallic and an asexual cycle represents the primary reproductive mode, C. reteaudii and C. auriculiformis are likely to be heterothallic and C. hongkongensis and C. aconidialis are homothallic. Based on the genetic diversity comparisons for C. pseudoreteaudii isolates from diseased leaves and soils, we hypothesize that C. pseudoreteaudii in soils was spread from diseased leaves. Both the mycelia plug and conidia suspension inoculations indicated that all five Calonectria species were pathogenic to the two Eucalyptus genotypes tested and the tolerance of the two genotypes differed. It is necessary to understand the ecological niche and epidemiological characteristics of these Calonectria species and to select disease resistant Eucalyptus genotypes in southern China in the future.

The genus Calonectria includes pathogens of various agricultural, horticultural, and forestry crops. Species of Calonectria are commonly collected from soils, fruits, leaves, stems, and roots. Some species of Calonectria isolated from soils are considered as important plant pathogens. Understanding the species diversity and distribution characteristics of Calonectria species in different soil layers will help us to clarify their long-term potential harm to plants and their patterns of dissemination. To our knowledge, no systematic research has been conducted concerning the species diversity and distribution characteristics of Calonectria in different soil layers. In this study, 1000 soil samples were collected from five soil layers (0–20, 20–40, 40–60, 60–80, and 80–100 cm) at 100 sampling points in one 15-year-old Eucalyptus urophylla hybrid plantation in southern China. A total of 1037 isolates of Calonectria present in all five soil layers were obtained from 93 of 100 sampling points. The 1037 isolates were identified based on DNA sequence comparisons of the translation elongation factor 1-alpha (tef1), β-tubulin (tub2), calmodulin (cmdA), and histone H3 (his3) gene regions, as well as the combination of morphological characteristics. These isolates were identified as C. hongkongensis (665 isolates; 64.1%), C. aconidialis (250 isolates; 24.1%), C. kyotensis (58 isolates; 5.6%), C. ilicicola (47 isolates; 4.5%), C. chinensis (2 isolates; 0.2%), and C. orientalis (15 isolates; 1.5%). With the exception of C. orientalis, which resides in the C. brassicae species complex, the other five species belonged to the C. kyotensis species complex. The results showed that the number of sampling points that yielded Calonectria and the number (and percentage) of Calonectria isolates obtained decreased with increasing depth of the soil. More than 84% of the isolates were obtained from the 0–20 and 20–40 cm soil layers. The deeper soil layers had comparatively lower numbers but still harbored a considerable number of Calonectria. The diversity of five species in the C. kyotensis species complex decreased with increasing soil depth. The genotypes of isolates in each Calonectria species were determined by tef1 and tub2 gene sequences. For each species in the C. kyotensis species complex, in most cases, the number of genotypes decreased with increasing soil depth. The 0–20 cm soil layer contained all of the genotypes of each species. To our knowledge, this study presents the first report of C. orientalis isolated in China. This species was isolated from the 40–60 and 60–80 cm soil layers at only one sampling point, and only one genotype was present. This study has enhanced our understanding of the species diversity and distribution characteristics of Calonectria in different soil layers.

The genus Calonectria includes many aggressive plant pathogenic species with a worldwide distribution. Calonectria leaf blight is one of the most prominent diseases of Eucalyptus trees in Southeast Asian and South American plantations. Inoculation trials to evaluate pathogenicity of Calonectria species typically use conidial suspensions but this is not possible for species that do not sporulate sufficiently in culture. Calonectria pseudoreteaudii is one of the most aggressive species to Eucalyptus in China, but most isolates fail to produce conidia in culture, requiring an alternative procedure for artificial inoculation. This study compared inoculations utilizing conidial and hyphal fragment suspensions. Two Eucalyptus genotypes were used, and these were inoculated with different concentrations of hyphal fragments or conidia of three C. pseudoreteaudii isolates. Three days after inoculation, the treated Eucalyptus plants displayed similar disease symptoms, irrespective of whether they had been inoculated with conidia or hyphal fragments. This was consistent for all of C. pseudoreteaudii isolates and also the different Eucalyptus genotypes. The results demonstrate that hyphal fragment suspensions can be used to provide a reliable indication of C. pseudoreteaudii isolate pathogenicity when conidia are not available for inoculation studies.

Fungi from Pseudofusicoccum (Phyllostictaceae, Botryosphaeriales) have been reported as pathogens, endophytes, or saprophytes from various woody plants in different countries. Recently, Botryosphaeriales isolates were obtained from the dead twigs of Acacia mangium, Eucalyptus spp., Pinus massoniana, and Cunninghamia lanceolata in Guangdong, Guangxi, Hainan, and Fujian Provinces in southern China. This study aimed to understand the diversity, distribution, and virulence of these Pseudofusicoccum species on these trees. A total of 126 Pseudofusicoccum isolates were obtained, and the incidences of Pseudofusicoccum (percentage of trees that yielded Pseudofusicoccum) on A. mangium, P. massoniana, Eucalyptus spp., and C. lanceolata were 21%, 2.6%, 0.5%, and 0%, respectively. Based on the internal transcribed spacer (ITS), translation elongation factor 1-alpha (tef1), and β-tubulin (tub2) loci, 75% of the total isolates were identified as P. kimberleyense, and the remaining isolates were identified as P. violaceum. For P. kimberleyense, the majority of isolates (83%) were from A. mangium, and the rest were from P. massoniana (14%) and Eucalyptus spp. (3%). Similarly, the proportion of isolates of P. violaceum from A. mangium, P. massoniana, and Eucalyptus spp. were 84%, 13%, and 3%, respectively. Inoculation trials showed that the two species produced expected lesions on the tested seedlings of A. mangium, E. urophylla × E. grandis, and P. elliottii. This study provides fundamental information on Pseudofusicoccum associated with diseases in main plantations in southern China.