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Fungus and insect collections
Surveys of diseased E. ingens were conducted in 2012 and 2013 at six sites with one inspection conducted at each site per year (Fig. 1; Sites 1 to 6). The same area, within which the belt transects were established for the symptom and mortality scoring survey, was used for the surveys at each site. At each site, one branch exhibiting each symptom type (grey discoloration of the succulent branches, rotting of the succulent branches surrounding moth damaged areas, or staining in the main woody stems associated with insect infestation) was collected from ten different trees for each symptom. Symptomatic tissue samples were placed in paper, and/or plastic bags and transported to the laboratory for further investigation.
Isolations for fungi were made by surface disinfesting plant tissue and cutting small segments from the leading edges of diseased areas and transferring the tissue to 2% malt extract agar (MEA; 15g agar and 20g malt extract l−1; Biolab, Merck, Midrand, South Africa) amended with streptomycin (Streptomycin; 0.4g l−1; Sigma-Aldrich, St Louis, USA). When fungal fruiting bodies were present on lesions or in insect tunnels, spore drops and/or hyphae were carefully removed from the plant material using a sterilized needle and placed on 2% MEA plates. The resultant colonies from tissues and fungal material were purified using single spore or hyphal tip transfers onto 2% MEA plates. After five days of growth, cultures were grouped according to each disease symptom and then further grouped based on the most commonly occurring pure cultures. Representatives from each morphological group were sequenced (using the ITS, LSU, β-tubulin and TEF 1-α gene regions) and identified to genus, and where possible, species level. Representative isolates have been deposited in the Culture Collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), Pretoria, South Africa.
Insects associated with diseased trees were obtained from freshly infested branches and stems by collecting 10 logs from 10 different trees from each site within the already established transect areas. Collections were made during March 2013 and 2014. For each site, four logs were placed in four emergence chambers which were monitored daily for insect emergence over a period of two weeks. Logs could not be kept for a longer period within the emergence chambers as E. ingens branches and stems rot and disintegrate very quickly due to their high moisture content. The remaining six logs, from each site, were dissected in the laboratory and insects collected pre-emergence. Insects collected from emergence chambers and dissected logs were grouped based on morphology using the keys in Wood (1986), and counts made for each group. Insects were identified by Dr. Roger Beaver (Thailand). Representative specimens of beetles were pinned and deposited with the National Collection of Insects, Plant Protection Research Institute, Agricultural Research Council, Roodeplaat, Pretoria, South Africa as well as the collection of the Tree Protection Co-operative Programme (TPCP), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa.
Estimation of mortality and disease symptoms associated with E. ingens die-off
Symptoms associated with die-off were present at all sites investigated and at varying levels of severity (Table 1). There was a significant difference in severity of the two main die-off symptoms (greying and rotting) as well as in the percentage mortality among the sites (Table 1). The sites with the highest proportion of rotting (moth damage) were Enzelsberg, Wolfaan, Ulundi followed by Bela-Bela, Euphorbia Drive, Last Post and Capricorn, Eshowe and Lydenburg. The sites with the highest proportion of grey discoloration were Euphorbia Drive, Last Post, and Ulundi followed by Capricorn, Eshowe, Lydenburg, Bela-Bela, Enzelsberg, and Wolfaan. Overall, the most severely affected sites (highest mortality) were Enzelsberg, Euphorbia Drive and Last Post with the least affected sites being Capricorn, Eshowe and Lydenburg. Sites with the highest mean rank greying did not always have the highest percentage of mortality of E. ingens and does not seem to be correlated (R2 = 0.01, P = 0.32). Euphorbia Drive, Last Post and Ulundi exhibited the highest mean rank greying with a correspondingly high percentage of mortality, while Capricorn had a high mean rank of grey discoloration with the lowest percentage mortality. Enzelsberg had the highest percentage mortality but the lowest mean rank of greying among all the sites. Moth damage was correlated (R2 = 0.212, P < 0.001) with higher percentage mortality, with the sites with the highest degree of die-off having higher levels of moth-related damage.
Fungus and insect collections
Isolations from diseased tissue yielded a total of 351 isolates for the six sites, with most isolates being saprophytes such as Penicillium species. From the 351 isolates, 100 were identified as the most consistently associated with the observed disease symptoms (Table 2). The isolates were divided into three main groups based on morphology. Representative isolates (Table 3) from each morpho-group were further identified using DNA sequence analysis, from which five genera were identified (Table 3).
Based on DNA sequence data, isolates were identified as Aureovirgo volantis (TreeBase: 17782, 17783) described previously by Van der Linde et al. (2016a) from E. ingens, an undescribed Fusarium sp. (TreeBase: 17784, 17785) in the Fusarium solani species complex, Lasiodiplodia×egyptiacae (TreeBase: 17788, 17789) (recently identified as a hybrid of L. theobromae and possibly L. parva or L. citricola; Cruywagen et al. 2016), Ophiostoma thermarum (TreeBase: 17782, 17783) described previously by Van der Linde et al. (2016a) from E. ingens, and an apparently undescribed Readeriella sp. (TreeBase: 17786, 17787). Lasiodiplodia×egyptiacae and F. solani were isolated from stained areas of the main stems of trees heavily infested with weevils as well as rotted tissues associated with moth damage. Readeriella sp. was isolated from fruiting bodies in grey as well as green succulent areas on the outside of the branches. Aureovirgo volantis and O. thermarum were commonly found within the tunnels of the ambrosia beetles Cyrtogenius africus and Stenoscelis sp., in succulent branches and the sapwood of the main stems (Fig. 2).
Fungal isolations were successful from only 55 (out of the 180 collected) branches (each branch from a different tree, N = 55 trees). Most of the isolates obtained were associated with insect damage (rotting associated with moth attacks and staining associated with weevil attacks) with only six isolates from greyed areas. Isolates associated with insect damage were obtained from all of the sites, while isolates from the grey discoloured tissue were obtained from only two sites (Table 2).
Five Curculionidae species (two ambrosia beetles and three other weevils) were collected from the emergence chambers (Table 2). The ambrosia beetles (Scolytinae), Cyrtogenius africus (AcP9546) and a Stenoscelis sp. (AcP9549), were reared from the main stems, while two weevils, Mechistocerus sp. (Molytinae) (AcP9551) and Coleobothrus germeauxi (Scolytinae) (AcP9544) were reared from the secondary phloem. The weevil Cossonus sp. (Cossoninae) (AcP9548) was reared from the vascular cambium (Fig. 3). Larvae of the moth Megasis sp. (Lepidoptera: Pyralidae) were identified at all sites and were associated with the rotting of the succulent branches.
Chapter 1
Literature review: Anthropogenic climate change effects on insects and pathogens affecting native and planted forests in the Northern Hemisphere and South Africa
Abstract
1. Introduction
2. The effect of anthropogenic climate change on tree insects and fungal pathogens
2.1 Insects
2.2 Pathogens
3. Anthropogenic climate change in southern Africa and its potential impact on tree health
3.1 Pathogens
3.2 Insects
4. Conclusions
5. Background and objectives of this thesis
6. References
Chapter 2
Fungi and insects associated with Euphorbia ingens die-off in South Africa
Abstract
1. Introduction
2. Materials and Methods
2.1 Estimation of mortality and disease symptoms associated with E. ingens die-off
2.2 Fungus and insect collections
3. Results
3.1 Estimation of mortality and disease symptoms associated with E. ingens die-off
3.2 Fungus and insect collections
4. Discussion
5. References
Chapter 3
Novel ophiostomatalean fungi from galleries of Cyrtogenius africus (Scolytinae) infesting dying Euphorbia ingens
Abstract
1. Introduction
2. Materials and Methods
2.1 Collection of samples and isolations
2.2 Fungal Morphology and Growth
2.3 DNA extraction, PCR, Sequencing and Phylogenetic analyses
2.4 Pathogenicity study
3. Results
3.1 Fungi isolated
3.2 Fungal Morphology and Growth
3.3 DNA sequence analyses Taxonomy
3.4 Pathogenicity
4. Discussion
6. References
Chapter 4
Seasonal flight patterns of Curculionidae (Cossoninae and Scolytinae) infesting dying Euphorbia ingens in South Africa
Abstract
1. Introduction
2. Materials and Methods
2.1 Study sites and collection of beetles
2.2 Temperature and relative humidity
2.3 Statistical Analyses
3. Results
3.1 Study sites and collection of beetles
3.2 Temperature and relative humidity factors
4. Discussion
5. References
Chapter 5
Landscape degradation may contribute to large-scale die-offs of Euphorbia ingens in South Africa
Abstract
1. Introduction
2. Materials and Methods
2.1 Study sites
2.2 Assessment of E. ingens mortality, degree of die-off, and the relationship of mortality and symptoms to climate and landscape variables
2.3 Data Analyses
3. Results
3.1 Levels and changes over time of Euphorbia ingens die-off symptoms and mortality
3.2 Precipitation and temperature
3.3 Euphorbia ingens mortality and landscape degradation
4. Discussion
6. References
Summary
