High genetic diversity and limited regional population differentiation in populations of Calonectria pseudoreteaudii from Eucalyptus plantations.

Calonectria pseudoreteaudii causes a serious and widespread disease known as Calonectria leaf blight in Eucalyptus plantations of southern China. Little is known regarding the population biology or reproductive biology of this pathogen in the affected areas. The aims of this study were to investigate the genetic diversity, population structure and the reproductive mode of C. pseudoreteaudii from affected Eucalyptus plantations of southern China. Ten polymorphic SSR markers were developed for the species, and were used to genotype 311 isolates from eight populations. The mating types of all isolates were identified using the MAT gene primers. The results revealed a high level of genetic diversity of the pathogen in all investigated populations. Of the 90 multilocus genotypes detected, ten were shared between at least two populations. With the exception of one population from HuiZhou, GuangDong (7HZ), the most dominant genotype was shared in seven remaining populations. DAPC and population differentiation analyses showed that the 7HZ population was well differentiated from the others and that there was no significant differentiation between the remaining populations. AMOVA suggested that most molecular variation was within populations (86%). Index of association analysis was consistent with a predominantly asexual life cycle for C. pseudoreteaudii in the studied regions. Although both mating types were detected in seven of the eight populations, the MAT1-1/MAT1-2 ratios in these populations deviated significantly from the 1:1 ratio expected in a randomly mating population.

Diversity of Calonectria species from leaves and soils in diseased southern China Eucalyptus plantation.

Calonectria leaf blight (CLB) is one of the best-known diseases of Eucalyptus spp., particularly in Asia and South America. Recently, typical symptoms of leaf and shoot blight caused by Calonectria spp. Were observed in a Eucalyptus plantation in the YunNan Province of southwestern China. Isolations were made from diseased leaves and top soil collected below the diseased trees to determine the causal agent of the disease and to consider the distribution characteristics of the Calonectria species. This resulted in 417 isolates, of which 228 were from leaves and 189 were from soil. Based on comparisons of DNA sequences for the act (actin), cmdA (calmodulin), his3 (histone H3), rpb2 (the second largest subunit of RNA polymerase), tef1 (translation elongation factor 1-alpha) and tub2 (ß-tubulin) gene regions, as well as morphological characteristics, 11 Calonectria species were identified. These included Calonectria aciculata (0.7 %), Ca. colhounii (1.2 %), Ca. eucalypti (10.6 %) and Ca. honghensis (43.2 %) in the Ca. colhounii species complex, and Ca. aconidialis (15.3 %), Ca. asiatica (9.8 %), Ca. hongkongensis (1.0 %), Ca. ilicicola (6.0 %), Ca. kyotensis (0.5 %), and Ca. yunnanensis (11.3 %) in the Ca. kyotensis species complex. In addition, a novel species, accounting for 0.5 % of the isolates, was discovered and is described here as Ca. dianii sp. nov. in the Ca colhounii species complex. Most (99.1 %) of the isolates collected from the leaves resided in the Ca. colhounii species complex and a majority (95.8 %) of those from the soils were in Ca. kyotensis species complex. These results suggest that Calonectria spp. in the Ca. colhounii species complex infecting leaves might be adapted to that niche and that those in the Ca. kyotensis species complex are better adapted to a soil habitat.

Wide Distribution of Teratosphaeria epicoccoides and T. destructans Associated with Diseased Eucalyptus Leaves in Plantations in Southern China.

Species of Mycosphaerellaceae and Teratosphaeriaceae represent over 40% of the fungi identified on eucalypt leaves worldwide. These include some important pathogens that mainly cause leaf blight and spot, and result in increasingly negative impacts on global commercial eucalypt industries. Eucalyptus plantations are commonly cultivated in southern China for solid wood and pulp products. However, the species diversity and geographic distribution of Mycosphaerellaceae and Teratosphaeriaceae, associated with diseased plantation Eucalyptus leaves in China, have not been clarified. In this study, we conducted the first systematic surveys and sample collections of Mycosphaerellaceae- and Teratosphaeriaceae-like fungi from diseased plantation Eucalyptus leaves in southern China. In total, 558 isolates were obtained from 59 sampled sites in five provinces. One isolate was isolated from each tree. According to the disease symptoms, conidia morphological characteristics, and DNA sequence comparisons of ITS, tef1 and tub2 gene regions. The 558 isolates were identified as Teratosphaeria epicoccoides (312 isolates; 55.9%) and T. destructans (246 isolates, 44.1%). Both species were widely distributed in the sampled regions in southern China. The genotypes of T. epicoccoides and T. destructans were determined based on ITS, tef1, and tub2 sequences. The results showed that multiple genotypes of each species of T. epicoccoides and T. destructans exist in China. Additionally, isolates with multiple genotypes were obtained in all five sampled provinces. These results suggest that both T. epicoccoides and T. destructans are not clonal. This study proved that both T. epicoccoides and T. destructans are dominant species and widely distributed on diseased Eucalyptus leaves in southern China. The wide geographic distribution and potential high genetic diversity pose challenges for the disease management of Teratosphaeria leaf blight and leaf spot in China.

Evidence of High Genetic Diversity and Differences in the Population Diversity of the Eucalyptus Leaf Blight Pathogen Calonectria pseudoreteaudii from Diseased Leaves and Soil in a Plantation in Guangxi, China.

Calonectria pseudoreteaudii is an important causal agent of Eucalyptus leaf blight in southern China. This pathogen causes Eucalyptus tree disease across numerous regions in southern China. In addition to diseased leaves, C. pseudoreteaudii has occasionally been isolated from soil in Eucalyptus plantations. The aim of this study was to clarify whether C. pseudoreteaudii causing Eucalyptus leaf blight in China is mainly clonally reproduced and to determine the potential spreading mechanism of C. pseudoreteaudii between diseased leaves and soil. To this end, 10 polymorphic microsatellite markers were analyzed to detect the genetic diversity of 97 C. pseudoreteaudii isolates from diseased leaves and soil in a Eucalyptus plantation in Guangxi Zhuang Autonomous Region, southern China. The analysis showed that the genetic diversity of the isolates from both the diseased leaves and soil was high. However, the gene and genotype diversity of the C. pseudoreteaudii isolates from diseased leaves were higher than those of the isolates from the soil. Moreover, all genotypes detected in the isolates from the soil were also found in the isolates from the diseased leaves. Structural analyses did not show clear population structures related to the population substrates of the diseased leaves or soil, and molecular variance analyses indicated that no significant genetic differentiation existed between the diseased leaf and soil populations. These results suggest that C. pseudoreteaudii in soil spreads from diseased leaves, and that an asexual cycle is the primary reproductive mode in both diseased leaf and soil populations. This is the first study on the genetic diversity and population structure of C. pseudoreteaudii. The high genetic diversity and spread pathways of this pathogen may pose challenges in controlling the disease. C. pseudoreteaudii from both diseased leaves and soils in Eucalyptus plantations needs to be carefully monitored for disease control and management.

Wide Distribution and Intraspecies Diversity in the Pathogenicity of Calonectria in Soil from Eucalyptus Plantations in Southern Guangxi of China.

Eucalyptus spp. are extensively cultivated in southern China because of their adaptability and versatile timber production. Calonectria leaf blight caused by Calonectria species is considered a major threat to Eucalyptus trees planted in China. The GuangXi Zhuang Autonomous Region is the provincial region with the largest distribution of Eucalyptus plantations in China. The present study aimed to expound the species diversity and pathogenicity of Calonectria isolates obtained from the soil of Eucalyptus plantations in GuangXi. A total of 188 Calonectria isolates were recovered from the soil located close to Eucalyptus trees, and the isolates were identified based on the DNA sequence comparisons of the four partial regions of the translation elongation factor 1-alpha (tef1), ß-tubulin (tub2), calmodulin (cmdA), and histone H3 (his3) genes. The isolates were identified as Calonectria aconidialis (74.5%), C. hongkongensis (21.3%), C. pseudoreteaudii (2.1%), C. kyotensis (1.6%), and C. chinensis (0.5%). The inoculation results indicated that 40 isolates representing five Calonectria species were pathogenic to the three Eucalyptus genotypes. Two inoculated experiments consistently showed that the longest lesions were produced by the isolates of C. aconidialis. Some isolates of C. aconidialis, C. hongkongensis, and C. kyotensis produced significantly longer lesions than the positive controls, but not the isolates of C. pseudoreteaudii or C. chinensis. These results indicated that Calonectria isolated from the soil may pose a threat to Eucalyptus plantations. Some Calonectria isolates of the same species differed significantly in their virulence in the tested Eucalyptus genotypes. The resistance of different Eucalyptus genotypes to Calonectria isolates within the same species was inconsistent. The inoculation results in this study suggested that many Calonectria isolates in each species had different levels of pathogenicity, and many Eucalyptus genotypes need to be tested to select disease-resistant Eucalyptus genetic materials in the future. The results of the present study enhance our knowledge of species diversity and the potential damage caused by Calonectria in the soil of Eucalyptus plantations. Our results also provide new insights into the breeding of disease-resistant Eucalyptus genotypes for controlling Calonectria leaf blight in China in the future.

Identification and pathogenicity of Aurifilum species (Cryphonectriaceae, Diaporthales) on Terminalia species in Southern China.

The family of Cryphonectriaceae (Diaporthales) contains many important tree pathogens and the hosts are wide-ranging. Tree species of Terminalia were widely planted as ornamental trees alongside city roads and villages in southern China. Recently, stem canker and cracked bark were observed on 2-6 year old Terminalianeotaliala and T.mantaly in several nurseries in Zhanjiang City, Guangdong Province, China. Typical conidiomata of Cryphonectriaceae fungi were observed on the surface of the diseased tissue. In this study, we used DNA sequence data (ITS, BT2/BT1, TEF-1α, rpb2) and morphological characteristics to identify the strains from Terminalia trees. Our results showed that isolates obtained in this study represent two species of Aurifilum, one previously described species, A.terminali, and an unknown species, which we described as A.cerciana sp. nov. Pathogenicity tests demonstrated that both A.terminali and A.cerciana were able to infect T.neotaliala and two tested Eucalyptus clones, suggesting the potential for Aurifilum fungi to become new pathogens of Eucalyptus.

Characterization of Pseudofusicoccum Species from Diseased Plantation-Grown Acacia mangium, Eucalyptus spp., and Pinus massoniana in Southern China.

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 ß-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.

Phylogeny, Morphology, Distribution, and Pathogenicity of Seven Calonectria Species from Leaf-Blighted Eucalyptus in HaiNan Island, China.

Leaf blight caused by Calonectria species constrains Eucalyptus trees in China. Calonectria leaf disease on Eucalyptus in China was first reported in HaiNan Island in 1985. No systematic investigation of Calonectria species associated with diseased Eucalyptus in HaiNan has been performed. To understand the species diversity, distribution, and pathogenicity of these Calonectria, 400 Calonectria isolates were obtained from 278 diseased Eucalyptus planted in 17 sites in five regions. All 400 isolates were identified by DNA sequences of translation elongation factor 1-alpha, ß-tubulin, calmodulin, and histone H3 gene regions and on morphology. Seven species, C. acaciicola (198 isolates), C. pseudoreteaudii (161 isolates), C. reteaudii (29 isolates), C. hawksworthii (6 isolates), C. hongkongensis (4 isolates), C. auriculiformis (1 isolate), and C. chinensis (1 isolate), were identified. This is the first report of C. acaciicola in China. C. acaciicola, C. pseudoreteaudii, and C. reteaudii belong to the C. reteaudii species complex and accounted for 97% of all isolates. The three species overlapped in vesicle shape, macroconidia size, and macroconidia septa number. Region significantly influenced C. acaciicola and C. pseudoreteaudii distribution. Representative isolates of C. acaciicola, C. pseudoreteaudii, C. reteaudii, and C. hawksworthii producing abundant macroconidia were used in conidial suspension inoculation on Eucalyptus seedlings; all were highly pathogenic to the two tested genotypes. The tolerances of two Eucalyptus genotypes were significantly different. This first systematic investigation of Calonectria species associated with Eucalyptus leaf blight in HaiNan will aid selection of disease-resistant genotypes for managing Eucalyptus leaf blight caused by Calonectria species in China.

Diversity and Distribution of Calonectria Species in Soils from Eucalyptus urophylla × E. grandis, Pinus massoniana, and Cunninghamia lanceolata Plantations in Four Provinces in Southern China.

The species of Calonectria include many notorious plant pathogens and are widely distributed around the world. Leaf blight caused by Calonectria species is considered one of the most prominent diseases in Eucalyptus plantations in China. Some Calonectria species isolated from soils in Eucalyptus plantations are highly pathogenic to inoculated Eucalyptus genotypes. In southern China, the plantation trees Cunninghamia lanceolata, Eucalyptus spp., and Pinus massoniana are always adjacently planted, especially in FuJian, GuangDong, GuangXi, and YunNan Provinces. The aim of this study was to understand the diversity and distribution of Calonectria in soils from plantations of different tree species in different geographic regions. Soil samples were collected from 12 sampling sites in Eucalyptus urophylla × E. grandis, P. massoniana, and C. lanceolata plantations in FuJian, GuangDong, GuangXi, and YunNan Provinces. Approximately 250 soil samples were collected from each sampling site, and a total of 2991 soil samples were obtained. A total of 1270 Calonectria isolates were obtained from 1270 soil samples. The 1270 isolates were identified based on DNA sequence comparisons of the partial gene regions of act, cmdA, his3, rpb2, tef1, and tub2. These isolates were identified as 11 Calonectria species: Calonectria aconidialis (69.50%), C. kyotensis (13.10%), C. hongkongensis (10.80%), C. ilicicola (2.50%), C. asiatica (2.36%), C. curvispora (0.31%), C. chinensis (0.24%), C. pacifica (0.24%), C. yunnanensis (0.16%), and C. canadiana (0.08%) in the C. kyotensis species complex and C. eucalypti (0.71%) in the C. colhounii species complex. The three dominant species, C. aconidialis, C. kyotensis, and C. hongkongensis, were widely distributed. The richness of Calonectria (percentage of soil samples that yielded Calonectria) in soils in the eastern regions (relatively humid regions) was higher than that in the western regions. The Calonectria richness of E. urophylla × E. grandis, P. massoniana, and C. lanceolata plantations decreased gradually. For each of the three dominant species, its richness in the eastern regions was generally higher than that in the western regions; the species richness was highest in E. urophylla × E. grandis plantations for C. aconidialis, while for each of C. kyotensis and C. hongkongensis, its species richness was highest in P. massoniana plantations. The genetic variation in C. aconidialis, C. kyotensis, and C. hongkongensis was more greatly affected by geographic region than by plantation tree species. This study expanded our understanding of the richness, species diversity, and distribution characteristics of Calonectria in soils from the plantations of different tree species in different geographic regions in southern China. Results in this study enhanced our understanding of the influencing characteristics of geographic region and tree species on the species and genetic diversity of soilborne fungi.

Calonectria queenslandica: Causal Agent of Eucalyptus Leaf Blight in Southern China.

Calonectria leaf blight caused by Calonectria spp. is among the most serious diseases affecting the health and sustainability of Eucalyptus plantations in southern China. Recent outbreaks of this disease in GuangDong Province prompted a need to identify the species involved. Typical symptoms of Calonectria leaf blight were observed on 2-year-old Eucalyptus urophylla × E. grandis trees in a plantation in the ZhaoQing region. In total, 38 Calonectria isolates were collected from 32 diseased trees. All isolates were identified using DNA sequence analyses of the translation elongation factor 1-α (tef1), ß-tubulin (tub2), calmodulin (cmdA), and histone H3 (his3) gene regions. Phylogenetic analyses revealed that Calonectria queenslandica was the dominant species, accounting for 81.6% of the isolates collected. Other species isolated included C. pseudoreteaudii (10.5%), C. reteaudii (5.3%), and C. aconidialis (2.6%). This is the first report of C. queenslandica in China and all isolates had identical sequences in all four gene regions. PCR amplification using primers targeting the MAT1-1-1 and MAT1-2-1 genes in all C. queenslandica isolates revealed that only the MAT1-2 idiomorph was present. The results suggest that C. queenslandica was introduced into the sampled area with very limited genetic diversity. Pathogenicity tests were conducted on two Eucalyptus genotypes widely planted in the GuangDong Province using isolates representing all species collected. The results showed that these species could all cause disease but the predominance of C. queenslandica on infected trees suggests that it is the major driver of the disease problem studied. Different Eucalyptus genotypes used in the pathogenicity tests differed in susceptibility to infection by the Calonectria spp. tested, providing opportunities to avoid leaf blight by deploying disease-tolerant planting stock.

Diversity and Distribution of Calonectria Species from Plantation and Forest Soils in Fujian Province, China.

To meet the growing demand for wood and pulp products, Eucalyptus plantations have expanded rapidly during the past two decades, becoming an integral part of the southern China landscape. Leaf blight caused by various Calonectria spp., is a serious threat to these plantations. In order to explore the diversity and distribution of Calonectria spp. in Fujian Province soils, samples were collected in Eucalyptus plantations and adjacent plantings of Cunninghamia lanceolata, Phyllostachys heterocycle and Pinus massoniana as well as in natural forests. Three hundred and fifty-three Calonectria isolates were recovered from soil samples and they were identified based on a comparison of multilocus DNA sequence data for the act (actin), cmdA (calmodulin), his3 (histone H3), rpb2 (the second largest subunit of RNA polymerase), tef1 (translation elongation factor 1-alpha) and tub2 (ß-tubulin) gene regions, as well as morphological characteristics. Six known taxa including Calonectria aconidialis, Ca. hongkongensis, Ca. ilicicola, Ca. kyotensis, Ca. pacifica, Ca. pseudoreteaudii and one novel species described here as Ca. minensis sp. nov. were identified. Of these, Ca. aconidialis and Ca. kyotensis were the most prevalent species, and found in eight and seven sites, and four and five forest types, respectively. Calonectria spp. were most abundant in soils from Eucalyptus stands, followed by P. heterocycle and natural forests. Relatively few species were found in the soils associated with Cunninghamia lanceolata and Pinus massoniana. The abundance of known Calonectria spp. suggests that these fungi have been relatively well sampled in Fujian. The results are also consistent with the fact that most Calonectria diseases are found on Angiosperm as opposed to Gymnosperm plants.

Comparison of Hyphal Fragments and Spores to Evaluate the Pathogenicity of the Eucalyptus Leaf and Shoot Pathogen Calonectria pseudoreteaudii.

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 spp. typically use conidial suspensions but this is not possible for species that do not sporulate sufficiently in culture. Calonectria pseudoreteaudii is one of the species that is most aggressive 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, regardless of whether they had been inoculated with conidia or hyphal fragments. This was consistent for all 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.

Calonectria in the age of genes and genomes: Towards understanding an important but relatively unknown group of pathogens.

The genus Calonectria includes many aggressive plant pathogens causing diseases on various agricultural crops as well as forestry and ornamental tree species. Some species have been accidentally introduced into new environments via international trade of putatively asymptomatic plant germplasm or contaminated soil, resulting in significant economic losses. This review provides an overview of the taxonomy, population biology, and pathology of Calonectria species, specifically emerging from contemporary studies that have relied on DNA-based technologies. The growing importance of genomics in future research is highlighted. A life cycle is proposed for Calonectria species, aimed at improving our ability to manage diseases caused by these pathogens.

Soil-borne Calonectria (Hypocreales, Nectriaceae) associated with Eucalyptus plantations in Colombia.

Eucalyptus spp. are widely planted in Colombia as an important component of a growing paper and pulp industry. Leaf and shoot blight caused by Calonectria spp. was one of the first disease problems to emerge in these plantations. A survey of Eucalyptus plantations in four forestry regions of Colombia during 2016 resulted in a large number of Calonectria isolates from soil samples collected in the understories of trees having symptoms of Calonectria leaf and shoot blight. The aim of this study was to identify and resolve the phylogenetic relationships for these isolates using DNA sequence comparisons of six gene regions as well as morphological characters. From a collection of 107 isolates, seven Calonectria species residing in three species complexes were identified. Two of these represented undescribed species, namely C.exiguispora sp. nov. and C.guahibo sp. nov. Calonectriaparvispora and C.spathulata were the most commonly isolated species, each of which accounted for approximately 30% of the isolates. The results suggest that Colombia has a wide diversity of Calonectria spp. and that these could challenge Eucalyptus plantation forestry in the future.

Species Diversity and Distribution Characteristics of Calonectria in Five Soil Layers in a Eucalyptus Plantation.

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), ß-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.

Population Diversity and Genetic Structure Reveal Patterns of Host Association and Anthropogenic Impact for the Globally Important Fungal Tree Pathogen Ceratocystis manginecans.

Species in the Ceratocystis manginecans complex are important fungal pathogens of plantation trees globally. The most important hosts include species of Eucalyptus, Acacia, Mangifera, and Punica. Despite their relevance and widespread occurrence, little is known regarding their population genetics and how this might relate to their host associations or geographic regions in which they occur. A global collection of 491 isolates representing the C. manginecans complex, from four different plant hosts and nine countries, were genotyped using microsatellite markers. Population genetic analyses using numerous tools were conducted to interrogate how their genetic diversity and structure might be affected by host or areas of occurrence. Results of genetic diversity studies showed that when grouping isolates into populations based on their host associations, the population on Eucalyptus was most diverse, and it also has a broad global distribution. When considering countries of origin as a basis for defining populations, the gene and genotypic diversity were highest in populations from China, Indonesia, and Brazil. In contrast, populations from Oman and Pakistan collected from Mangifera had the lowest genetic diversity and were clonal. Molecular variance, population differentiation, and network and structure analyses showed that the genetic structure of isolates in the C. manginecans complex is influenced by both host association as well as geographical isolation. Furthermore, the results reflected the movement of genotypes between plant hosts and geographic regions that have implications regarding the broad global distribution of this pathogen.

Species Diversity, Mating Strategy and Pathogenicity of Calonectria Species from Diseased Leaves and Soils in the Eucalyptus Plantation in Southern China.

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), ß-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.

Global Genetic Diversity and Mating Type Distribution of Calonectria pauciramosa: An Important Wide-Host-Range Plant Pathogen.

The fungal pathogen, Calonectria pauciramosa, has caused serious diseases of many important plants worldwide. Understanding the genetic diversity and mating type distribution of this pathogen provides an essential step toward the development of disease control measures. In this study, we designed 15 polymorphic microsatellite markers by using genome sequences of two Ca. pauciramosa isolates having opposite mating type and from different countries. These markers were used to determine the genetic diversity of 145 isolates representing 13 different hosts (12 plant hosts residing in 12 genera, and soil) from 10 countries. In addition, mating type genes were amplified to investigate the reproduction mode of the pathogens in these populations by using mating type primers designed for Calonectria spp. Results revealed that a single dominant genotype, isolated from 11 plant genera residing in eight families, was present in seven countries across five continents. Only mating type MAT1-1 or MAT1-2 was amplified in each of the isolates, confirming that Ca. pauciramosa is heterothallic. Both mating types were detected in isolates from Eucalyptus in South Africa and Uruguay. The MAT1-2 phenotype was widely distributed in isolates from 12 different hosts (11 plant hosts and soil) collected in 10 countries. Overall, the results suggest that there has been substantial global movement of Ca. pauciramosa and that this has shaped its current population structure.

Variation in Botryosphaeriaceae from Eucalyptus plantations in YunNan Province in southwestern China across a climatic gradient.

The Botryosphaeriaceae accommodates many important pathogens of woody plants, including Eucalyptus. Recently, Botryosphaeriaceae were isolated from diseased plant parts from surveys of Eucalyptus plantations in the YunNan Province, China. The aims of this study were to identify these Botryosphaeriaceae isolates and to evaluate their pathogenicity to Eucalyptus. A total of 166 isolates of Botryosphaeriaceae were obtained from six regions in the YunNan Province, of which 76 were from Eucalyptus urophylla × E. grandis hybrids, 49 from E. globulus trees, and 41 isolates were from other unknown Eucalyptus species or hybrids. Isolates were identified by comparing DNA sequences of the internal transcribed spacer ribosomal RNA locus (ITS), partial translation elongation factor 1-alpha (tef1), ß-tubulin 2 (tub2) and DNA-directed RNA polymerase II subunit (rpb2) genes, and combined with their morphological characteristics. Eleven species were identified, including Botryosphaeria fusispora, B. wangensis, Lasiodiplodia pseudotheobromae, Neofusicoccum kwambonambiense, N. parvum, and six novel species described as B. puerensis, N. dianense, N. magniconidium, N. ningerense, N. parviconidium and N. yunnanense. The dominant species across the regions were N. yunnanense, N. parvum and B. wangensis, representing 31.3, 25.3 and 19.9% of the total isolates, respectively. Species diversity and composition changed across the different climatic zones, despite their relatively close geographic proximity and the fact that some of the species have a global distribution. All the Botryosphaeriaceae species were pathogenic to one-year-old plants of an E. urophylla × E. grandis clone and E. globulus seed-derived plants, but showed significant inter- and intra-species variation in aggressiveness amongst isolates. The study provides a foundation for monitoring and management of Botryosphaeriaceae through selection and breeding of Eucalyptus in the YunNan Province of southwestern China.