Needle Fungi in Young Tasmanian Pinus Radiata Plantations in Relation to Elevation and Rainfall Istiana Prihatini1,2, Morag Glen1*, Timothy J
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Prihatini et al. New Zealand Journal of Forestry Science (2015) 45:25 DOI 10.1186/s40490-015-0055-6 RESEARCH ARTICLE Open Access Needle fungi in young Tasmanian Pinus radiata plantations in relation to elevation and rainfall Istiana Prihatini1,2, Morag Glen1*, Timothy J. Wardlaw3, David A. Ratkowsky1 and Caroline L. Mohammed1 Abstract Background: Needle fungi in conifers have been extensively studied to explore their diversity, but environmental factors influencing the composition of fungal communities in Pinus radiata D.Don needles have received little attention. This study was conducted to examine the influence of the environment as defined by rainfall, elevation and temperature on the composition of fungal communities in pine needles at an age prior to that at which spring needle cast (SNC) is generally observed. Elucidating the entire fungal community in the needles is a first step towards understanding the cause of the disease. Methods: Needle samples were collected from 5-year-old P. radiata trees, their age predating the onset of SNC, from 12 plantations in Tasmania. Interpolated data for the climate variables, including seasonal components for rainfall and temperature, were obtained from an enhanced climate data bank. Pooled needle samples were examined for the fungi they contained using DNA sequencing of cloned polymerase chain reaction (PCR) products. Clones were grouped into operational taxonomic units (OTUs) and identified to their lowest possible taxonomic level by comparison with reference isolates and public DNA databases. Results: DNA sequencing revealed that needle fungal communities differed greatly, depending upon the total annual rainfall and needle age. Needle fungi that have been previously associated with pathogenic behaviour, such as Cyclaneusma minus, Dothistroma septosporum, Lophodermium pinastri, Strasseria geniculata and Sydowia polyspora, were all found in the needles in this study. Also, of 70 ascomycetous OTUs that were discriminated by their DNA sequences, 21 were identified as belonging to the Teratosphaeriaceae. Conclusions: An overview of the endophytic fungi present in the needles of P. radiata has been obtained from trees whose age precedes the onset of SNC. All of the previously known pathogenic species for needle cast diseases were present, in addition to a range of unidentified OTUs belonging to the Teratosphaeriaceae. Keywords: Needle fungi, Pinus radiata, Rainfall, Elevation, Direct PCR Background 2007). These factors can affect the complex interactions As described in previous studies (Ganley & Newcombe between microbial organisms, which in turn influence 2006; Koukol et al. 2012; Terhonen et al. 2011), the their interactions with their host plant (Giauque & Pinus phyllosphere (i.e. the environment for microorgan- Hawkes 2013). Abiotic or environmental factors influ- isms on and below the needle surface) is a rich and var- encing the composition of fungal communities associ- ied microbial community. Its composition is strongly ated with Pinus radiata D.Don needles have received influenced by both genotypic and environmental factors little attention. (Botes et al. 1997; Van Maanen et al. 2000; Wang & Guo Studies have shown that endophyte communities in Pinus sylvestris L. needles can vary according to geo- graphic location (Gourbière & Debouzie 2003) and * Correspondence: [email protected] 1Tasmanian Institute of Agriculture, University of Tasmania, Private Bag 98, elevation (Gourbière et al. 2001). Communities in Picea Hobart, Tasmania 7001, Australia mariana (Mill.) Britton, Sterns & Poggenb. needles have Full list of author information is available at the end of the article © 2015 Prihatini et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Prihatini et al. New Zealand Journal of Forestry Science (2015) 45:25 Page 2 of 10 been reported to vary with latitude (Sokolski et al. 2007). and Wardlaw (1990) found that 23 of the sites were A study by Hata and Futai (1996) showed that the endo- visually assessed as having SNC, and that these stands phytic mycota associated with 45 pine species or varieties were restricted to a mean annual rainfall range of was similar for those pines belonging to the same taxo- 1200–2000 mm and mean annual temperature range of nomic group. That study also concluded that endophytic 8–11.5 °C. Podger and Wardlaw (1990) putatively asso- colonisation might reflect the degree of adaptation of the ciated Cyclaneusma minus, Lophodermium pinastri and host to local biotic and abiotic factors. However, consider- Strasseria geniculata with the disease in Tasmania, but able controversy exists about whether an endophyte, usu- there were many areas with severe SNC where Podger ally an organism such as a bacterium or a fungus, can and Wardlaw (1990) found no fungal fruiting bodies of become a pathogen. Some authors have suggested that en- these species on attached needles, dead or alive. How- dophytes may play a role in protecting trees from patho- ever, these three species of fungi were ubiquitous on gens (Arnold et al. 2003; Bradshaw 2004; Gao et al. 2010), fallen needles at sites both affected and unaffected by but others maintain that a commensal or mutualist fun- SNC. More recently, fungal communities were surveyed gal species can be triggered under certain environmen- in a P. radiata family trial established in 1999 in Oonah, tal conditions to act as a virulent parasite (Krabel et al. Tasmania. The trial was assessed at 8 years of age in 2007 2013). The definition of an endophyte differs greatly, using DNA extraction from needles, polymerase chain but in a comprehensive chapter on endophytic fungi, reaction (PCR) with fungal-specific primers and sequencing Stone et al. (2004) wrote that, in general, the term to detect the presence of fungal species (Prihatini et al. endophytic fungi applies to ‘fungi capable of symptom- 2015a). Trees with contrasting levels of SNC disease sever- less occupation of apparently healthy plant tissue’. ity were found to have significantly different needle fungal Broadly, this means that endophytes are viewed as colo- communities. The four fungi, L. pinastri, C. minus, S. geni- nising living plant tissues without causing them any im- culata and Dothistroma septosporum,previouslyreported mediately obvious negative effect. Nevertheless, others, as common pathogens or endophytes in P. radiata in such as Brown et al. (1998), who elucidated the endo- Tasmania implicated in causing SNC (Podger 1984; Podger phytic fungal communities of the Musa acuminata and Wardlaw 1990) were also identified in the Prihatini et Colla species (banana) complex and found 24 taxa of al. (2015a) study. Of these, only L. pinastri was corre- which almost all were ascomycetes, raised the question lated with high levels of disease. Several species of ‘Can endophytes of Musa spp. become pathogens?’ Teratosphaeriaceae were also detected. Teratosphaeria- They noted the strong correlation between lists of ceae sp. 23 was detected in almost all of the needle pathogenic taxa of Musa spp. and frequently isolated samples and was presumed to be endophytic as it was tropical endophyte genera, but in the context of their associated with needles from healthy trees as well as study, they determined that it was not possible to con- those affected by SNC. In contrast, Teratosphaeriaceae clude if banana pathogens isolated as species of endo- sp. 3 was detected most frequently in needles from se- phytes in healthy leaves were the same pathogenic verely diseased trees and not at all in needles from trees strains. As the fungi may be non-pathogenic strains, with <5 % disease. An overview of the fungal species they concluded that experiments following Koch’spos- detected in these two studies (Prihatini et al. 2015b) tulates were required to confirm any pathogenicity. highlighted the discrepancy between fungal associates Similarly, Larran et al. (2002) and Douanla-Meli et al. of P. radiata detected by PCR and sequencing and (2013) found that their lists of fungal species in the those detected by isolation. healthy leaves of soybean and citrus, respectively, were The present study was conducted in parallel with predominantly or almost exclusively made up of com- Prihatini et al. (2015a, b) to explore the types and diversity mon plant pathogens, but each study concluded that of needle fungi present in 5-year-old P. radiata in Tasmania, further investigations were necessary to understand the an age at which all trees have green, non-symptomatic precise role of these endophytes. needles, and to examine the influence of climate and land- Spring needle cast (SNC) in Tasmania appears in pine form as defined by rainfall, elevation and temperature plantations at canopy closure (age 6–7 years) and is on the composition of fungal communities. The motiv- manifested by the rapid browning of 1-year-old needles ation for this was to obtain a catalogue of fungal spe- followed by the heavy casting of needles. Spring needle cies from healthy tissues that could point towards the cast is not considered to be a classic needle blight dis- fungi that caused, or might be implicated in, SNC. This ease caused by a primary fungal pathogen (Podger & follows the approach of Petrini (1986) and Carroll Wardlaw 1990); instead, it is thought to be due to a suite (1988), who advocated systematic sampling of healthy of endophytic fungi that are triggered into secondary plant tissue for internal fungi rather than the usual pathogenic activity by an environmental stress. In their practice of the time of examining diseased or necrotic study of 53 stands selected for climatic analysis, Podger plant organs. Prihatini et al. New Zealand Journal of Forestry Science (2015) 45:25 Page 3 of 10 Methods the same class were pooled for each site, placed into Sampling sites and environmental data paper bags and stored at 10 °C for 1–2 days before Twelve P.