Anna Maria Vettraino

Pyrosequencing of environmental soil samples reveals biodiversity of the Phytophthora resident community in chestnut forests

Pyrosequencing analysis was performed on soils from Italian chestnut groves to evaluate the diversity of the resident Phytophthora community. Sequences analysed with a custom database discriminated 15 pathogenic Phytophthoras including species common to chestnut soils, while a total of nine species were detected with baiting. The two sites studied differed in Phytophthora diversity and the presence of specific taxa responded to specific ecological traits of the sites. Furthermore, some species not previously recorded were represented by a discrete number of reads; among these species, Phytophthora ramorum was detected at both sites. Pyrosequencing was demonstrated to be a very sensitive technique to describe the Phytophthora community in soil and was able to detect species not easy to be isolated from soil with standard baiting techniques. In particular, pyrosequencing is an highly efficient tool for investigating the colonization of new environments by alien species, and for ecological and adaptive studies coupled with biological detection methods. This study represents the first application of pyrosequencing for describing Phytophthoras in environmental soil samples.

Ectomycorrhizal community structure in a healthy and a Phytophthora -infected chestnut ( Castanea sativa Mill.) stand in central Italy

Ink disease caused by Phytophthora cambivora is a major disease of sweet chestnut (Castanea sativa). In two C. sativa stands in central Italy, one (Montesanti) that is infected with P. cambivora and the trees showing symptoms of ink disease and another healthy stand (Puzzella), the ectomycorrhizal (ECM) community structure was investigated. On the roots of the surviving trees of the diseased stand, 29 different ECM species were determined compared to 23 in the healthy stand. Eleven ECM species were common to both stands; however, a number of species were unique to one of the stands. Cenococcum geophilum was dominant at both sites, but the percentage colonisation was much higher at Montesanti (40.8%) compared to Puzzella (27.2%). There was a switch in species from Russula vesca, Russula lepida and Russula azurea at Puzzella to Russula nigricans, R. lepida and Russula delica at Montesanti. Both R. vesca and R. azurea were found only at the Puzzella site. At the diseased site, the ECMs formed had a smaller root tip diameter, and the ECM at the healthy site had more abundant extramatrical hyphae.

Phytophthora himalsilva sp. nov. an unusually phenotypically variable species from a remote forest in Nepal

The Himalaya have received little investigation for Phytophthora species. In a remote forest in Western Nepal ten isolates of an unknown Phytophthora were recovered from the rhizosphere of Quercus, Castanopsis, Carpinus and Cupressus spp. The Phytophthora, formally named here as a P. himalsilva sp. nov., is homothallic with either amphigynous or paragynous antheridia and papillate, highly variable sporangia which may also be facultatively caducous. Based on ITS, β-tubulin, and cox I sequences Phytophthora himalsilva falls within Phytophthora Clade 2c together with Phytophthora citrophthora, Phytophthora meadii, Phytophthora colocasiae, and Phytophthora botryosa. It is suggested that Clade 2c has radiated within Asia. Molecular and sporangial characters indicate that P. himalsilva and P. citrophthora may share a recent common ancestor although they have diverged in their breeding systems. Although highly local the P. himalsilva isolates exhibited significant variation in growth rates and optimum temperatures for growth. This may reflect adaptation to different niches within a heterogeneous sub-tropical to temperate forest environment. Their cox I polymorphisms were also rather variable, including possible clustering for subsite. The occurrence of a previously unknown Phytophthora in a remote forest in Nepal highlights once again the plant health risk associated with moving rooted plants and soil between different bio-geographical regions of the world and the need for rapid pathological screening of potential risk organisms.

Endophytism of Sclerotinia pseudotuberosa: PCR assay for specific detection in chestnut tissues

The endophytic behaviour of Sclerotinia pseudotuberosa, cause of black rot of fruits of Castanea sativa, was investigated in asymptomatic tissues of the host, including nuts, buds and bark. A nested polymerase chain reaction (PCR) assay was developed for the specific detection of S. pseudotuberosa. Two specific primers, RAC1 and RAC2 were designed within the ITS1-5.8S-ITS2 region of rDNA by comparison of sequences of 24 isolates of S. pseudotuberosa and related species. Specificity of primers was tested against isolates of related and unrelated genera and the total DNA of C. sativa. The assay proved to be highly sensitive and capable of detecting picograms of S. pseudotuberosa DNA. Our study showed that S. pseudotuberosa was present asymptomatically in all the chestnut tissues tested. It is discussed that latency of S. pseudotuberosa in chestnut tissues may represent a adaptive strategy of the pathogen for rapid and massive host colonization in favourable environmental conditions.

Four phenotypically and phylogenetically distinct lineages in Phytophthora lateralis.

Until recently Phytophthora lateralis was known only as the cause of dieback and mortality of Chamaecyparis lawsoniana in its native range in the Pacific Northwest (PNW). Since the 1990s however disease outbreaks have occurred increasingly on ornamental C. lawsoniana in Europe; and in 2007 the pathogen was discovered in soil around old growth Chamaecyparis obtusa in Taiwan, where it may be endemic. When the phenotypes of over 150 isolates of P. lateralis from Taiwan, across the PNW (British Columbia to California) and from France, the Netherlands and the UK were compared three growth rate groups were resolved: one slow growing from Taiwan, one fast growing from the PNW and Europe, and one of intermediate growth from a small area of the UK. Within these growth groups distinct subtypes were identified based on colony patterns and spore metrics and further discriminated in a multivariate analysis. The assumption that the three main growth groups represented phylogenetic units was tested by comparative sequencing of two mitochondrial and three nuclear genes. This assumption was confirmed. In addition two phenotype clusters within the Taiwan growth group were also shown to be phylogenetically distinct. These four phenotypically and genotypically unique populations are informally designated as the PNW lineage, the UK lineage, the Taiwan J lineage, and the Taiwan K lineage. Their characteristics and distribution are described and their evolution, taxonomic, and plant health significance is discussed.

The sentinel tree nursery as an early warning system for pathway risk assessment: Fungal pathogens associated with Chinese woody plants commonly shipped to Europe

Introduction of and invasion by alien plant pathogens represents the main cause of emerging infectious diseases affecting domesticated and wild plant species worldwide. The trade in living plants is the most common pathway of introduction. Many of the alien tree pathogens recently introduced into Europe were not previously included on any quarantine lists. To help determine the potential risk of pest introduction through trading of ornamental plants, a sentinel nursery was established in Beijing, China in 2008. The sentinel nursery planting included four of the most common ornamental woody species shipped to Europe including Ilex cornuta var. fortunae, Zelkova schneideriana, Fraxinus chinensis and Buxus microphylla. Symptoms developing on these species within the sentinel nursery were detected in 2013 and consisted of necrotic spots on leaves, canker and stem necrosis, shoot blight and shoot necrosis. Fungi associated with the trees and their symptoms included Alternaria alternata detected from all hosts; Diaporthe liquidambaris and Diaporthe capsici from bark and leaf necrosis of Zelkova schneideriana; Botryosphaeria dothidea and Nothophoma quercina from stem cankers on Fraxinus chinensis and leaf necrosis on Ilex cornuta; and Pseudonectria foliicola from leaf necrosis on Buxus microphylla. Next generation sequencing analysis from asymptomatic tissues detected eighteen OTU’s at species level among which some taxa had not been previously recorded in Europe. These results clearly demonstrate that looking at trees of internationally traded species in the region of origin can reveal the presence of potentially harmful organisms of major forestry, landscape or crop trees. Results of this study also provide an indication as to how some disease agents can be introduced using pathways other than the co-generic hosts. Hence, sentinel nurseries represent one potential mechanism to address the current lack of knowledge about pests in the countries from where live plants are shipped and the threats they represent to native flora and crops in importing countries.

Manufactured wood trade to Europe: a potential uninspected carrier of alien fungi

Sets of mangrove wood utensils shipped to Italy from China were found to be colonised by a number of fungal taxa. The fungi were found proliferating and sporulating on utensil surfaces after despatch to the market. The same taxa have been found to be present as asymptomatic infections in inner tissues. Most of the taxa were not pathogenic to plants, whereas one of the taxa was an isolate of the plant pathogenic species Fusarium oxysporum. The data presented in this manuscript provide clear evidence of how the hidden movement of fungi by the unrestricted trade in uninspected woody products should be considered a common and high risk pathway of invasion.

International plant trade and biosecurity

This chapter explores the current status of plant trade and international biosecurity regulatory mechanisms to safeguard economic, social and economic well being of nations, states and economic regions. We provide an account of the international biosecurity framework in a historical context. In doing so we outline some of the common approaches to managing and regulating biosecurity risks associated with the plant horticultural export trade. This exploration identifies many of the inconsistencies in the application of plant biosecurity measured internationally. The approaches for regulation of live plants are compared amongst regions and future improvements are identified.

Castanea sativa Mill. (Avrupa kestanesi)‘ da Phytophthora

Avrupa (veya tatli) kestanesi ( Castanea sativa Mill.) Avrupa’da Castanea cinsinin tek dogal turudur. C. sativa ’nin dogal dagilim sinirlari muhtemelen Anadolu’da Karadeniz boyunca Kafkasya’ya kadar uzanan bolgede yer almaktadir. Şu anda, bu tur Bati Avrupa’dan doguya kadar (http://www.discoverlife.org), yillik toplam yagisin 600 mm den daha yuksek oldugu yerlerde, hafif asidik topraklarda (pH 4.5-6.5) oldukca yaygin dagilim gostermektedir (Urbisz & Urbisz, 2007). Avrupa’da, C. sativa ’ kulturu uzun zamanli bir gelenektir (Conedera et al. , 2004). Bu tur, yenilebilir meyveleri ve ideal teknolojik ozellikli mukemmel odunu ile kirsal alan icin buyuk bir ekonomik degere sahiptir. Bundan da ote, kestane koruluklari onemli bir agro-ekolojik rol oynamaktadir, ornegin yangina ve erozyona karsi koruma, yaban hayati icin habitat ve rekreasyon alanlari gibi. Ticari anlamda kestane uretimi icin, Avrupa ve Japon kestaneleri ( C. crenata ) arasindaki melezler dahil farkli cesitler (kulturu yapilan varyeteler) gelistirilmistir. Cesitler bir cok ozellikleri bakimindan, ornegin, buyukluk ve meyvelerin organoleptik ozellikleri dahil, farklilik gosterirler.

Interaction between pathogens and water in disease development in agriculture and forest ecosystems

The present paper reports on the role of water in plant pathology as possible stress factor and vector of pathogen. The latter aspect is considered in a scenario of general risk of introduction and spread of invasive plant pathogens. In addition to peculiar epidemiology aspects, the possible diagnostic methodologies and control methods are considered. The role of water as stress factor is analysed in a general frame of climatic global changes that could enhance the risk of severe drought events. Within this frame some model pathosystems are described where water plays a role as co-factor or inciting factor in disease development.