Population structure analysis provides insights into the infection biology and invasion strategies of Kretzschmaria deusta in trees (original) (raw)

Abstract

Infection biology and invasion strategies of Kretzschmaria deusta were investigated through the analysis of patterns of colonization and population diversity in three case studies, comprising different host species. Molecular analysis and isolation assays performed on stem sections at different heights indicated a prevalent heart rot mode of expansion. Random amplified microsatellites – RAMs and somatic incompatibility assays on isolates allowed detection, in one case, of a genet occupying the entire decay column and, in the other cases, of several different genets in each individual tree. Hypotheses on modes of arrival and entrance of K. deusta in trees are discussed on the basis of the distribution of genets and population genetics analyses. Significant correlation (Spearman ρ = 1.0; p < 0.001) between the number of genets and the number of areas delimited by pseudosclerotial plates (PSPs) on the decayed portions of stem sections suggests PSPs are interaction zone lines between different individuals of K. deusta occupying adjacent decay columns.

Highlights

► Kretzschmaria deusta has a heart rot mode of establishment in beech, maple and plane trees. ► Kretzschmaria deusta may infect trees via airborne spores through wounds, as suggested by the population structure analysis. ► Pseudosclerotial plates can be the result of interaction among different individuals of Kretzschmaria deusta.

Introduction

Although often regarded merely as a saprotroph, the xylariaceous ascomycete Kretzschmaria deusta is locally reported as a primary root rot, butt rot, trunk rot and canker rot of several tree species (Hawksworth 1972; Sinclair et al. 1987). In tropical and subtropical regions, K. deusta can pose a threat for commercial plantations, e.g. oil palms, red grapefruit, rubber and tea trees (Sinclair et al. 1987; Palacios et al. 2008), whereas in temperate regions it is reported as one of the main reasons for decreased safety and windthrows of Acer spp., Aesculus spp., Fagus spp., Tilia spp. and Ulmus spp. trees (Wilkins 1934, 1939; Greig 1989; Gibbs & Greig 1990; Terho & Hallaksela 2008; Michelotti et al. in press). Sudden limb failures are also often associated with K. deusta (Lonsdale 1999). The detection of K. deusta during tree inspection is often problematic, even at an advanced stage of colonization. Indeed, signs of its presence, mainly consisting of inconspicuous conidial and perithecial stromata, can easily be missed during visual tree inspections. Even common instrumental analysis, including those based on penetrometers and acoustic diagnostic devices, can often fail to detect wood decay of K. deusta, which acts predominantly as a soft rot during tree colonization (Schwarze et al. 1995; Brandstetter 2007).

As a consequence, most studies on K. deusta have focused either on advanced techniques for the detection of decay (Pearce et al. 1994; Schwarze et al. 1995; Rabe et al. 2004; Deflorio et al. 2008a) and its early identification in trees (Nicolotti et al. 2009), or on the dynamics of fungal invasion and host response through artificial inoculation assays (Baum et al. 2000; Schwarze & Baum 2000; Deflorio et al. 2008b, 2009). Based on the results of these studies, the micro-morphological features of the brittle decay caused by this ascomycete and aspects concerning its colonization strategy, i.e. mechanisms of reaction zone penetration and ability to colonize sapwood of certain tree species, have been exhaustively elucidated.

Conversely, although parasitism of K. deusta towards several tree species was demonstrated and deeply investigated in earlier case studies performed by Wilkins (1936, 1938, 1939, 1943), no clear evidence defining its infection biology, i.e. the modes of spread and entrance, has been reported so far. Such evidences can be provided by studies on the genetic structure of pathogen populations within and among hosts. The abundance, size and distribution of different genets (genetic individuals) may be considered as an indirect reflection of fungal mode of spread, entrance and establishment among and within the trees (Rayner & Boddy 1986). Briefly, vegetative mycelium through root contacts or soil provides the opportunity for one genet to spread from tree-to-tree, whereas arrival by propagules, i.e. sexual and asexual spores, may give rise to a more heterogeneous population. Similarly, within a tree, population diversity is expected to be higher where infection took place. In fact, during decay progress many genets can be filtered out due to spatial competition for domain and/or selectivity of the environment (Rayner & Boddy 1986). Analysis of population structure can further be useful to improve knowledge of the role of pseudosclerotial plates (PSPs) often characterizing the colonization of K. deusta in trees. Although these crust-like aggregations of melanised hyphae are commonly reported as barriers to maintain a dry moisture regime favourable for K. deusta growth (Rayner & Boddy 1988), no studies have been performed to test the hypothesis that PSPs may be interaction zone lines between decay columns occupied by different individuals.

The presence of different genets within a population can be detected by testing for somatic incompatibility (SI) between vegetative mycelia (Lane 1981). This method has allowed determination of intraspecific diversity in populations of xylariaceous fungi both on a local scale, i.e. within single ascomata or trees and among adjacent trees in a woodland site (Chapela & Boddy 1988; Griffin et al. 1992; Rodrigues et al. 1995; Hendry et al. 1998; Johannesson et al. 2001), and on a larger scale, i.e. throughout wide geographical areas (Pérez Jiménez et al. 2002). Molecular methods based on randomly amplified polymorphic DNA (RAPD) markers proved to be even more effective in discriminating genets within fungal populations (Jacobson et al. 1993). While the SI assays can reveal the genetic differences associated with the loci involved in the self/non-self recognition, molecular genotyping can access genome-wide polymorphisms (Douhan et al. 2011). Although reproducibility of RAPD markers is often questionable, a variant of this technique, based on random amplification of DNA regions flanked by microsatellites (random amplified microsatellites – RAMs or inter simple sequence repeats – ISSR), allows more reproducible fingerprints (Gente et al. 2002) and has proved to be a valuable tool for assessing genetic diversity within populations of parasitic, saprotrophic and mutualistic symbiotic fungi (Vainio et al. 1998; Gherbi et al. 1999; Paavolainen et al. 2001).

The present study was aimed at investigating the infection biology and invasion strategies, i.e. modes of arrival, entrance and establishment, of K. deusta in three case studies, each involving a different tree species located either in urban environment or in a forest site. Each case study was thus focused on: (i) assessing the patterns of colonization of K. deusta in trees through both isolation assays and multiplex PCR (M-PCR)-based molecular analysis performed on wood samples collected from stem sections at different heights and on soil samples surrounding trees; (ii) determining the population structure of K. deusta through RAMs analysis and SI assays within each tree and stem section, and among trees in one site. Results of population structure analysis have further been used to test the hypothesis that PSPs are the result of mycelial interaction between different individuals occupying adjacent decay columns.

Section snippets

Tree material and sampling procedures

The three case studies comprised four trees in which K. deusta had been previously detected through a drill-based technique combined with M-PCR-based molecular analysis (Guglielmo et al. 2010). The first case included an Acer platanoides (ID: A1) and an Acer pseudoplatanus (A2) tree located in the Turin city area 15 m apart along the same street (45°03′40″N; 7°39′22″E); the second case consisted of a Fagus sylvatica tree (F) located in the forest of the nature reserve of Sacro Monte di Varallo,

Isolation assays and M-PCR analysis

A total of 387 wood samples were analyzed (Table 1). K. deusta was detected through isolation assays in 85 samples and through M-PCR in further 120 samples. Most of the wood samples positive for K. deusta (203 out of 205) were collected from areas of stem section with evidence of decay and surrounding hollows (Table 1). K. deusta was detected in only two of the 103 samples excised from discoloured areas bordering the decayed areas. None of the samples collected from branches and from

Discussion

The three case studies conducted in this work, and focused on patterns of colonization and population structure of K. deusta within trees, provided evidence supporting a prevalent heart rot mode of expansion and suggested different modes of arrival and entry, presumably linked to environmental determinants. The identification of different genets in stem sections indicates PSPs can be the result of interactions between genets of K. deusta occupying adjacent decay columns.

A common feature of all

Acknowledgements

This study was supported by a grant of the Municipality of Torino – Divisione Ambiente – Settore Verde Pubblico. We are grateful to the Nature Reserve of Sacro Monte di Varallo and to the Municipality of Cirié. We also thank the anonymous reviewers for helpful comments on the manuscript.

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