Botryosphaeriaceae from Acacia karroo in South Africa

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Chapter 2 Greater Botryosphaeriaceae diversity in healthy than associated diseased Acacia karroo tree tissues

ABSTRACT

Botryosphaeriaceae are common endophytes of trees. Some species are also known to be pathogens. It is, therefore, assumed that endophytic Botryosphaeriaceae are often involved in general die-back diseases. Here we test this assumption in severe branch die-back observed on Acacia karroo trees in the Pretoria area of South Africa. The presence of the Botryosphaeriaceae was compared between healthy and diseased tissue on the same trees. Eight Botryosphaeriaceae species were isolated from die-back and healthy branches. Of these, six species, namely Tiarosporella urbis-rosarum, Diplodia allocellula, Phaeobotryosphaeria variabilis, Dothiorella brevicollis and Neofusicoccum vitifusiforme were obtained from healthy tissues, and only two species, Dothiorella dulcispinae and Spencermartinsia pretoriensis, were exclusively found in die-back branches. Spencermartinsia viticola was found in both tissue types and this fungus was also the most commonly isolated species from both healthy and die-back samples. Results of pathogenicity trials showed highly variable results for the isolated species and that the two species associated only with die-back symptoms, were weakly pathogenic. These results suggest that the Botryosphaeriaceae found in these trees were not directly associated with the die-back symptoms, despite their diversity and common occurrence in these tissues. The situation is different in other tree systems where dominant species, often with wide host ranges, have been shown to be involved in die-back diseases. This indicates the importance of characterizing the unique aspects of each tree disease system.

INTRODUCTION

Species residing in the fungal family Botryosphaeriaceae include latent pathogens that occur asymptomatically as endophytes for extended periods, but cause disease under stress conditions (Slippers and Wingfield 2007; Smith et al. 1996; Van Niekerk et al. 2011; Denman et al. 2000). Symptoms of these diseases include die-back followed by resin exudation, blackish discoloration of the heartwood and pith, fruit rot, leaf blight, premature leaf drop, gummosis and in severe cases tree death (Slippers et al. 2007; Slippers and Wingfield 2007). Some species of the Botryosphaeriaceae have wide host ranges and they also occur on all continents other than Antarctica (Slippers and Wingfield 2007; Taylor et al. 2009).
The pathogenicity of some Botryosphaeriaceae species has been well established, but the true role of most described species in disease is poorly studied. Many Botryosphaeriaceae have been isolated from die-back symptoms, others only from asymptomatic tissues and some have been found in both tissue types (Slippers and Wingfield 2007). When tested, many species have been shown to be pathogenic (Slippers and Wingfield 2007). For instance, species from die-back symptoms that have been shown to be aggressive pathogens in artificial inoculation trials include Diplodia africana on Juniperus phoenicea (Linaldeddu et al. 2012), Neofusicoccum parvum on Eucalyptus globulus (Iturritxa et al. 2011) and Syzygium paniculatum (Ploetz et al. 2009), Lasiodiplodia theobromae, L. pseudotheobromae and L. egyptiacae on Mangifera indica (Ismail et al. 2012) and Botryosphaeria dothidea, N. luteum, N. mediterraneum and N. parvum on Ficus microcarpa (Mayorquin et al. 2012). However, L. gonubiensis has been isolated from asymptomatic tissues of the native tree S. cordatum (Pavlic et al. 2004), but was also shown to cause lesions in pathogenicity trials (Pavlic et al. 2007). Several species have been isolated from both healthy and die-back tissues. Lasiodiplodia theobromae were isolated from necrotic branches of Vaccinium species (Wright and Harmon 2009), vine die-back (Taylor et al. 2005; Van Niekerk et al. 2004) and healthy tissues of Terminalia catappa (Begoude et al. 2010) and Eucalyptus spp. (Pérez et al. 2010), while L. margaritacea were isolated from both healthy and die-back symptoms on native Adansonia gregorii (Pavlic et al. 2008; Sakalidis et al. 2011). All of these species were shown to be pathogenic in pathogenicity tests. The pathogenicity of some other Botryosphaeriaceae isolated from die-back symptoms, such as N. protearum from die-back of native Protea spp. (Denman et al. 2003), Spencermartinsia viticola and Dothiorella iberica on grapevine in New South Wales and South Australia (Luque et al. 2005; Pitt et al. 2010) still remain unknown in plant pathogenicity tests. This matter is further complicated because many studies fail to clearly indicate whether isolates have been obtained as endophytes or from diseased tissue.
Acacia karroo or sweet thorn (Fabales: Mimosoideae) is the most widespread native Acacia in southern Africa (Timberlake et al. 1999) and plays an important role in increasing soil fertility through nitrogen fixation with rhizobia nodules (Barnes et al. 1996). Die-back symptoms on branches of A. karroo are common in South Africa but they have become quite severe in the Pretoria area (Gauteng Province). Larval tunnels of an unidentified cerambycid beetle were sometimes observed in these dieback symptoms, and especially in the necrotic parts of the branch samples (Figure 1). Cerambycid beetles have been reported from Acacia species in various parts of the world (Eisa and Roth 2009; Elliott and De Little 1985; Watt 1983). The larvae of cerambycid beetles (Coleoptera: Cerambycidae) are xylophagous and create a network of tunnels while feeding in different tissues of healthy, dead or decaying woody tissues of plants (Haack and Slansky 1987). Some cerambycid beetles can directly kill the trees or branches because their feeding in the cambium layers destroys the vascular tissues (Rad 2006; Hawkeswood 2011). These larvae could thus be involved in causing some of these symptoms or cause stress to the tree, but were not associated with them frequently enough to be the main causal agent.
Botryosphaeriaceae species are known to be associated with die-back symptoms on A. karroo and other Acacia trees in South Africa (Jami et al. 2012; Van der Walt 2008), and could thus be associated with the increased die-back of A. karroo in the Pretoria area. The aim of this study was to determine whether these species of Botryosphaeriaceae are also present and associated with the striking branch die-back symptoms in Pretoria, which have not been sampled previously. Due to the endophytic nature of the Botryosphaeriaceae, species of these fungi would most likely be associated with branches. Species occurring in die-back branches were thus compared with those found in asymptomatic tissues to establish a better understanding of the diversity of species existing as endophytes on these trees, and their potential relationship with those involved in the die-back symptoms.

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MATERIALS AND METHODS

Collection of samples

Samples were collected from 40 A. karroo trees having branches with die-back at different locations in the greater Pretoria area, Gauteng Province. The die-back begins where leaves on branch tips begin to wilt, turn brown and die, but they remain attached to the‎ plant‎ resulting‎ in‎ “flagging”‎ symptoms (Figure 1). Other than wilting, no symptoms were observed on the leaves. Lesions were formed in the woody tissue, with dead tissue extending internally within branches and often associated with gum production on the outside of the branches. These lesions were clearly the cause of the die-back symptoms as the wilting only occurred to the terminal ends of these lesions and wilting due to problems at the roots were unlikely.
A single branch showing die-back with internal lesions and an asymptomatic branch were collected from each tree. The branch samples were placed in paper bags and transported to the laboratory. For endophyte isolations, a selected portion of each branch was cut into 0.5 cm and twelve pieces were randomly selected from each branch. From the die-back branches, the portions selected were taken from the border zone between healthy and discolored wood. All of these amounted to 480 pieces in total from die-back and 480 from healthy branches, which were surface-disinfested in 10 % hydrogen peroxide for two minutes, and rinsed three times in sterile water. Representative samples from all branches were placed onto 2 % malt extract agar (four pieces per plate) (Biolab, Midrand, South Africa). Pieces from diseased tissue were thus also placed onto agar selective for Phytophthora (NARPH) (Shearer and Dillon 1995) and between two slices of carrot for the isolation of Ceratocystis species (Moller and Devay 1968).
The plates and carrot pieces were incubated at 24 °C for seven days and the fungal growth from each wood sample showing morphology characteristic of the Botryosphaeriaceae was transferred from the primary isolations to new MEA plates. After 4-5 days, all those isolates showing typical fast growing, white to black cultures with fluffy aerial hyphae were transferred to 15% WA (water agar) in order to make single hyphal tip sub-cultures. These isolates are maintained in the Culture Collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa.

DNA sequence analyses

Isolates (Table 1) were initially grouped based on culture morphology (fast growing, white to black cultures with aerial hyphae). DNA was extracted from the mycelium of 5-day-old single hyphal-tip cultures (Lee and Taylor 1990) representing three cultures for each morphological group. Sequence data were obtained for the internal transcribed spacer region of the ribosomal RNA (rRNA) operon using primers ITS-1 (Gardes and Bruns 1993) and ITS-4 (White et al. 1990), the‎β-tubulin gene using primers Bt2a and Bt2b (Glass and Donaldson 1995), the translation elongation factor 1-α‎(TEF-1α)‎ gene using primers EF1-728F and EF1-986R (Carbone and Kohn 1999) and the large subunit rDNA (LSU) gene region using primers LR0 and LR5 (Vilgalys and Hester 1990).

Acknowledgments
Preface
Chapter 1 Five new species of Botryosphaeriaceae from Acacia karroo in South Africa
Chapter 2 Greater Botryosphaeriaceae diversity in healthy than associated diseased Acacia
karroo trees tissue
Chapter 3 The pattern of Botryosphaeriaceae on four unrelated native South African hosts
Chapter 4 Botryosphaeriaceae associated with Acacia karroo in South Africa: Temporal and
spatial variation
Chapter 5 Diversity and distribution of the Botryosphaeriaceae in South Africa and Namibia
Summary

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