Improving disease resistance of butternut (Juglans cinerea), a threatened fine hardwood: a case for single-tree selection through genetic improvement and deployment

Approaches for the development of disease-resistant butternut (Juglans cinerea L.) are reviewed. Butternut is a threatened fine hardwood throughout its natural range in eastern North America because of the invasion of the exotic fungus, Sirococcus clavigignenti-juglandacearum Nair, Kostichka and Kuntz, which causes butternut canker. Early efforts were made to identify and collect putatively resistant germ plasm, identify vectors and to characterize the disease. More recently, molecular techniques have been employed to genetically characterize both the pathogen and the resistant germ plasm. Much of the host resistance may originate from hybridization with a close Asian relative, Japanese walnut (Juglans ailanthifolia Carr.), and from a few natural phenotypic variants. Further genetic characterization is needed before classical breeding or genetic modification can be used to produce canker-resistant trees.     

Recent insights into the pandemic disease butternut canker caused by the invasive pathogen Ophiognomonia clavigignenti‐juglandacearum

In the 25 years following the initial 1967 report of the disease, butternut canker was able to quickly spread throughout the entire range of butternut (Juglans cinerea) in North America, from Minnesota in the upper Midwest to Tennessee in the south and Quebec in the north‐east. The speed of this dispersal is notable as butternut trees do not make up a significant proportion of any single forest type. Instead, they are usually found sparingly in most mixed hardwood forests. In this review, we synthesize the current knowledge of the invasion process of the butternut canker pathogen, Ophiognomonia clavigignenti‐juglandacearum, an invasive fungal pathogen, that as its emergence has spread across North America and is now found wherever butternut naturally occurs. Taxonomic studies have determined that the fungus belongs in the genus Ophiognomonia, which includes a number of saprophytes, endophytes and pathogens of members of the Fagales, rather than the genus Sirococcus, which includes several important pine pathogens. The ability of fungus to be dispersed by rain splash, transported on and in beetle vectors, transmitted by infected seed and successfully colonized several species of Juglans and Carya have all likely contributed to the rapid increase in abundance and severity of disease and tree mortality in the invaded forest ecosystems. Recent genomic and population genetic analyses have determined that there were at least three emergence events. A less virulent strain of the fungus likely has been present in the north eastern United States for over a century, but it was the emergence of a more virulent strain of the fungus in Minnesota and Wisconsin in the 1960s that resulted in range‐wide mortality and pushed butternut to be listed as an endangered species in Canada and a number of states in the United States.     

qPCR quantification of Ophiognomonia clavigignenti‐juglandacearum from infected butternut trees under different release treatments

The use of a molecular assay for quantifying conidia of Ophiognomonia clavigignenti‐juglandacearum, the fungal pathogen responsible of butternut canker, was investigated. Purified DNA from conidia collected on glass fibre filters of a passive rain collectors was quantified using a TaqMan real‐time quantitative polymerase chain reaction (qPCR) assay. The qPCR assay could specifically discriminate the target species from all other North American known species of Ophiognomonia, and it was sensitive enough to repeatedly detect one conidium. A linear relationship between numbers of conidia and qPCR C_t values was determined, and used to assess the sporulation of the pathogen under trees that were released to promote their vigour. In total, 977 samples of field‐captured conidia from 49 trees, at two locations, and from two successive growing seasons were analysed. No significant difference of sporulation was observed under control and release treatments. However, our results demonstrated that qPCR assay was reliable for detecting and quantifying O. clavigignenti‐juglandacearum from environmental samples, which will be useful to assess further control methods for this disease.     

Susceptibility of Eucalyptus hybrid clones to Botryosphaeria canker in Uganda

ABSTRACT

The study assessed the susceptibility of the nine commonly grown Eucalyptus clones to Neofusicoccum species associated with Botryosphaeria canker in Uganda. The inoculation trials indicated that susceptibility of Eucalyptus hybrids differed significantly (p = .000), clones GU609, GU7, GC578, and GC796 exhibiting a higher tolerance than GC784, GC550, GU8, GC514, and GC540. The results further revealed that N. parvum was more pathogenic than N. kwambonambiense. The generated information can be exploited in sustainable forest management by expanding the growing of tolerant hybrids in areas with high Botryosphaeria canker disease pressure.     

Diseases on Eucalyptus species in Zimbabwean plantations and woodlots

Zimbabwe embarked on planting Eucalyptus species in the early 1900s. Based on a robust breeding programme, it has become a major source of seed for other countries in and outside Africa. Tree health surveys conducted on Eucalyptus in some east and southern African countries over the past two decades have revealed several important fungal diseases that were previously not known in the region, but little is known regarding these problems in Zimbabwe. The aim of this study was to identify important Eucalyptus diseases across Zimbabwe's agroclimatic regions. Morphological characteristics and DNA sequence data were used to identify pathogens collected to species level. Widespread stem canker diseases, caused by species belonging to the Botryosphaeriaceae and Teratosphaeriaceae, and leaf spot diseases caused by fungi in the Capnodiales, were identified. Armillaria root and stem rot was restricted to a single site in the Eastern Highlands. Fungi that could cause canker or blue stain of timber were isolated from recently harvested stumps and included species of Ceratocystis and Ophiostoma. This study is the first to identify Eucalyptus pathogens to species level in Zimbabwe and we report for the first time the presence of the stem canker pathogen T. gauchensis in southern Africa. The results will provide a foundation for the formulation of future disease management strategies in the country.     

中国柳属次级区分的探讨(二)

中国柳属约有200个种（以及大量的杂种和一些外来种）。本文中，该属被区分为38个组。以种为一整体的概念编制了分组检索表，详细记述了每一个组，同时列出了全部的种类（舍杂种和外来种），包括它们的名称（正名、异名）、地理分布等。     

Endophytic foliar fungi in Betula spp. and their F1 hybrids

We examined foliar endophyte frequencies in two native (Betula pendula and Betula pubescens) and three exotic (Betula ermanii, Betula platyphylla and Betula resinifera) birch species and their hybrids in Punkaharju, Finland. The most frequently isolated endophytic fungi in the experimental trees were Fusicladium betulae and Gnomonia setacea making up 80–90% of all endophyte infections. Total endophyte infection levels varied from 0.5 colony forming units (CFU)/cm² in B. platyphylla to 8.6 CFU/cm² in B. pubescens that had highest total infection levels of both examined endophyte species. The resistance of hybrids was generally very close to the more resistant parent (the only exception being Fusicladium in B. platyphylla × B. pendula hybrid) supporting the hypothesis that the resistance of birch hybrids to these fungi is genetically based and caused by dominant inheritance of resistance traits.     

中国柳属次级区分的探讨(一)

中国柳属约有200个种（以及大量的杂种和一些外来种）.本文中,该属被区分为38个组,以种为一整体的概念编制了分组检索表,详细记述了每一个组,同时列出了全部的种类（含杂种和外来种）,包括它们的名称（正名、异名）、地理分布等.     

Botryosphaeriaceae species causing canker and dieback of English walnut (Juglans regia) in Italy

English walnut (Juglans regia) is an important nut crop worldwide and is currently considered emerging in Italy. Botryosphaeriaceae fungi cause symptoms including cankers, discoloration and dieback, and several species are reported across the world on walnut. In this study, symptomatic trees from an orchard in Southern Italy showing branch dieback, cankers, wood discoloration and gummosis were surveyed. Three different fungi from the Botryosphaeriaceae were consistently isolated from symptomatic tissues. Representative isolates were characterized using morphological and molecular approaches based on conidial morphology, optimum growth temperature and the comparison of DNA sequence data from the ITS, tef1‐α and tub2 loci. Three species were identified: Botryosphaeria dothidea, Neofusicoccum mediterraneum and Neofusicoccum parvum. Pathogenicity tests on detached fruits and potted plants showed that all three species were pathogenic. To our knowledge, this is the first report of these Botryosphaeriaceae species causing canker and dieback on English walnut in Italy.     

河北省葡萄座腔菌科菌物系统分类研究

【目的】葡萄座腔菌科菌物是引起众多经济及生态造林树种枝干溃疡、枝枯类病害的病原生物,在世界各地均有广泛的分布。本研究对河北省内,与经济林木病害相关的葡萄座腔菌科菌物进行系统分类研究,目的在于揭示该科菌物在特定地区的地理分布特征,明确该类菌物所致树木枝干溃疡、枝枯类病害的寄主植物范围,为河北省树木溃疡、枝枯类病害的控制提供理论依据。【方法】在对河北省树木溃疡类病害系统调查的基础上,本研究根据菌物无性型及有性型的形态学特征、rDNA-ITS序列特征对包括河北省石家庄在内的8个市(县、区)25个不同地点的葡萄座腔菌科菌物进行分类鉴定和系统分类。【结果】共获得葡萄座腔菌科菌物5种73株,分别属于Botryosphaeria dothidea、B.stevensii、B.obtusa、B.sarmentorum和Phyllosticta citrichinaensis。其中B.dothidea为河北省葡萄座腔菌科优势菌物种,占全部检出菌物菌株总数的77%。葡萄座腔菌科菌物侵染杨树、柳树、苹果、核桃等28种植物,其中包括大叶黄杨、构树、火炬树、连翘、花椒和刺梅等6种新寄主植物。【结论】B.dothidea不仅危害杨树等传统寄主,而且危害花椒等新寄主植物以及入侵树种火炬树,是危害河北省树木健康生长的最为重要、分布最为广泛的枝干病原菌物,有必要对其溃疡致病性分化和种群遗传分化进行深入分析。来自柿树、花椒等寄主的10个具有Dothiorella型分生孢子的菌株形成单系类群,提示这些菌株可能是与B.sarmentorum和B.iberica不同的一个系统发育学新种。     

The first record of Botryodiplodia canker in Poland

This study describes the first observation of Botryodiplodia canker in the Western Carpathians in south‐eastern Poland caused by Botryodiplodia hypodermia (Sacc.) Petr. (syn. Sphaeropsis hypodermia, S. ulmicola). The canker occurred on an approximately 17‐year‐old Ulmus glabra sapling in a mixed conifer/deciduous stand with elm trees severely damaged by Dutch elm disease. This paper describes disease symptoms and provides information on the macro‐ and micromorphology of the fungus isolated from the cankered tissues. The results of BLAST search using DNA sequences obtained for our cultures and subsequent phylogenetic positioning of the fungus among closely related Botryosphaeriaceae indicate that the species is much more closely related to Phaeobotryon than to the other Botryodiplodia or Sphaeropsis species. Moreover, a total of 16 polymorphisms within the ITS region were detected between S. ulmicola associated with Botryodiplodia canker in North America and B. hypodermia associated with the canker observed in Poland. Thus, the “European” variant of “Sphaeropsis” ulmicola can now be easily identified with our barcode sequences. The Botryodiplodia canker is much less prevalent in Europe than in North America. Differences in virulence of “American” and “European” linages and differences in susceptibility of various elm species may be the reason for the higher prevalence of the disease in North America.     

杨树溃疡病潜伏侵染的研究

在陕西省杨树溃疡病(Dothiorella gregaria Sacc.)是一种严重的病害,分布普遍,危害130多个杨树品种、杂交种和无性系,其中有些树种如箭杆杨、北京杨、陕林2号杨、箭×钻杨等受害更为严重,常导致整株死亡,造成重大损失。据研究杨树溃疡病菌有潜伏侵染的特性,但在陕西省杨树苗木上未见其发病,有无潜伏侵染现象存在,更不清楚,为了弄清这一问题,我们于1987—1988年,对来自三个苗圃的24种杨树苗木     

Plant somatic hybrid cytoplasmic DNA characterization by single-strand conformation polymorphism

Unlike maternal inheritance in sexual hybridization, plant somatic hybridization allows transfer, mixing and recombination of cytoplasmic genomes. In addition to the use of somatic hybridization in plant breeding programs, application of this unique tool should lead to a better understanding of the roles played by the chloroplastic and mitochondrial genomes in determining agronomically important traits. The nucleotide sequences of cytoplasmic genomes are much more conserved than those of nuclear genomes. Cytoplasmic DNA composition in somatic hybrids is commonly elucidated either by length polymorphism analysis of restricted genome regions amplified with universal primers (PCR-RF) or by hybridization of total DNA using universal cytoplasmic probes. In this study, we demonstrate that single-stranded conformational polymorphism (SSCP) analysis is a powerful, quick and easy alternative method for cytoplasmic DNA characterization of somatic hybrids, especially for mitochondrial DNA. The technique allows detection of polymorphisms based on both size and sequence of amplified targets. Twenty-two species of the subfamily Aurantioideae were analyzed with eight universal primers (four from chloroplastic and four from mitochondrial regions). Differences in chloroplastic DNA composition were scored in 98% of all possible two-parent combinations, and different mitochondrial DNA profiles were found in 87% of them. Analysis by SSCP was also successfully used to characterize somatic hybrids and cybrids obtained by fusion of Citrus sinensis (L.) Osb. and C. excelsa Wester protoplasts.     

全球松树枯梢病发生状况与防治策略

松树枯梢病是一种世界性病害。该病寄主范围广,可侵染8个属约60种（含变种）针叶树。对全球松树枯梢病为害较严重国家（新西兰、南非、美国和中国等）的发生状况以及近年有关该病发生发展的流行诱因和抗性研究等进行了概述,并提出以树种生态适应性、提高寄主生长势为主以及控制病菌增量的防治策略。     

树木溃疡病病原真菌类群分子遗传多样性研究Ⅱ--Botryosphaeria属28S rDNA-PCR-RFLP和RAPD解析

BotryosphaeriaCes.etdeNot .(无性阶段是DothiorellaSacc .)属病原真菌能引起多种林木溃疡病的发生 ,在我国分布区域十分广泛。病原菌因较大的寄主范围和地理分布区域表现出明显的分子遗传分化 ,通过对来自 5个区 1 4个寄主种 (品种 )的 30个菌株进行 2 8SrDNA PCR RFLP和RAPD解析 ,结果表明 ,所有供试菌株都能扩增出稳定一致的 2 8SrDNA条带 ,6种内切酶不能区分该属内部的 2 8SrDNA差异。RAPD结果表现出该属丰富的多样性 (92 81 % ) ,在 0 845相似系数的水平上所有菌株被识别为 1 3类 ,其中有地理分化、寄主分化的表现 ,也有不表现地理、寄主分化的。从DNA水平上证实引起雪松溃疡病的病原为B .dothidea ,表明B .dothidea不仅能够寄生被子植物 ,还能寄生裸子植物 ,有非常宽阔的寄主范围。随机扩增引物K1 6和R1 4可以作为陕西菌株和湖南菌株与其它地区菌株区别的鉴别性引物。传统上认为苹果轮纹病菌 (B .berengerianadeNot.f.sp .piricola (Nose)KoganezawaetSakuma)为苹果干腐病菌 (B .berengerianabeNot.)的专化型 ,它们与杨树溃疡病原分属两个种。本试验分析表明这种分类较为合理。     

Testing of selected South African <Emphasis Type="Italic">Pinus</Emphasis> hybrids and families for tolerance to the pitch canker pathogen, <Emphasis Type="Italic">Fusarium circinatum</Emphasis>

Plantations of Pinus spp. constitute approximately 50% of the South African forestry industry. The first aim of this study was to develop a reliable inoculation technique to screen Pinus spp., for tolerance to infection by F. circinatum, which threatens pine forestry in South Africa. Inoculation of branches was compared with stem inoculations and we considered the number of branches or trees required to obtain statistically significant results. Furthermore, variation in the susceptibility of some Pinus families, clones and hybrids was considered. Results showed that branch inoculations were closely correlated with those from stem inoculations, and that it is important to consider branch and stem diameters when assessing susceptibility of trees. Subsequent trials using branch inoculations showed significant differences in F. circinatum tolerance amongst a range of pine species and hybrids of potential interest to forestry in South Africa. Significant differences in susceptibility were also found among clones of two P. radiata families. The most tolerant trees were P. elliottii × caribaea and P. patula × oocarpa hybrids, while the most susceptible species were P. patula, P. greggii and hybrids of these two. This is the first trial considering the susceptibility of Pinus hybrids, Pinus clones and some P. patula provenances, and the results indicate excellent potential for breeding for tolerance to pitch canker in South Africa. Application The accurate selection of disease tolerant planting stock for the South African forestry industry is crucially important for the continued sustainability of this important industry. The work described here provides valuable information on an artificial inoculation technique that will assist the industry in screening trees for tolerance to the pitch canker fungus, F. circinatum. It also provides some indication of the relative susceptibility of a number of Pinus spp., hybrids and families currently being evaluated in the country.     

Variation in susceptibility of six pine species and hybrids to pitch canker caused by Fusarium circinatum

Six pine species or hybrids were tested for susceptibility to pitch canker caused by Fusarium  circinatum. Pinus  densiflora, Pinus  thunbergii, Pinus  x rigitaeda (Pinus  rigida × Pinus  taeda), P. rigida × P. x rigitaeda, Pinus  echinata and Pinus  virginiana were inoculated with three spore loads (50, 500 and 5000 per tree) of F. circinatum. External symptoms, lesion length, and the frequency of reisolation of the fungus were investigated. External symptoms were greatest in P. echinata, followed by P. virginiana, however, P. densiflora was not susceptible to F. circinatum. Based on mean lesion lengths, the six pine species or hybrids differed significantly (p < 0.01) in susceptibility to pitch canker. Pinus  echinata sustained the longest lesions, whereas P. densiflora sustained the shortest lesions. The effect of inoculum density was not significant among three spore treatments within species (p = 0.17), although lesion length was slightly greater at higher spore loads over all pine species. The fungus was reisolated from inoculated stems of all pine species tested, even on trees showing little or no damage from the disease. Additional studies are needed to further explore the basis for resistance to pitch canker.     

Pezicula canker of Quercus rubra L., caused by Pezicula cinnamomea (DC.) Sacc.

The ascomycete Pezicula cinnamomea (DC.) Sacc. [Anamorph: Cryptosporiopsis grisea (Pers.) Petr.] causes Pezicula canker of Red Oak (Quercus rubra L.) in Germany and is also associated with bark diseases of other oak species. The author's studies on morphology, biology and en-dophytism of P. cinnamomea are presented and its ecological role is discussed. It can be con-cluded that P. cinnamomea is a species with a broad host range. The fungus is a common en-dophyte of Red Oak bark, and development of Pezicula canker is dependent on reduction of host vigour.     

A severe canker disease of Corymbia ficifolia caused by Quambalaria coyrecup in native and urban forests of Western Australia

A serious canker disease is affecting the health of Corymbia ficifolia in urban areas of Perth, Western Australia. Quambalaria coyrecup was frequently associated with diseased trees and its presence confirmed by morphological characters and DNA sequencing. A number of Quambalaria species have previously been identified as causal agents of canker and shoot blight diseases on a range of Eucalyptus and Corymbia species. It was therefore the aim, using glasshouse studies, to determine whether Q. coyrecup was the primary causal agent of the cankers on C. ficifolia and whether other Quambalaria species could also be associated with the disease. All seedlings inoculated with Q. coyrecup produced canker symptoms within 1 month after inoculation. Canker lesions typical of those observed in the field also occurred in the four subsequent months, after which time the trial was terminated. Inoculation with Q. cyanescens and Q. pitereka (isolated from C. ficifolia shoots) did not result in lesion development. This study establishes that Q. coyrecup is a serious pathogen of C. ficifolia and that wounding is required for canker development to occur. The management of Quambalaria canker must therefore include the minimization of artificial wounding of trees in the nursery and field.