BIOLOGY AND ECOLOGY OF CERATOCYSTIS SPECIES WITH AN EMPHASIS ON THEIR INSECT ASSOCIATIONS

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Chapter 2 Ceratocystis species on Acacia mearnsii and Eucalyptus spp. in eastern and southern Africa including six new species

ABSTRACT

Species of Ceratocystis include well-known plant pathogens causing cankers, vascular wilt and root diseases, as well as many species that are agents of sap stain of lumber. A number of Ceratocystis spp. have been reported from Africa, but the continent is  generally poorly sampled in terms of these fungi. The  aim of this study was to consider the presence of Ceratocystis spp. infecting wounds on plantation-grown, non-native, Acacia mearnsii and Eucalyptus spp. in Kenya, Malawi, Tanzania and South Africa. Isolates were collected from cut stumps and artificially induced wounds on the stems of trees. They were subsequently identified based on morphological characteristics  and  DNA sequence comparisons for the ribosomal RNA Internal Transcribed Spacer region, including the 5.8S operon as well as partial sequences of the β-tubulin and the Transcription Elongation Factor 1α genes. Analyses showed that isolates represent six previously undescribed species from Malawi, South Africa and Tanzania, while C. moniliformis was found for the first time in  Tanzania.  The  undescribed  Ceratocystis spp. are provided with the names C. zombamontana prov. nom., C. polyconidia prov. nom., C. tanganyicensis prov. nom., C. obpyriformis prov. nom., C. oblonga prov. nom. and T. ceramica prov. nom. The wilt  pathogen C. albifundus was also commonly found on A. mearnsii in Tanzania and in Kenya.  All the new species described in this study  were pathogenic on the hosts from which they were originally isolated.

INTRODUCTION

The genus Ceratocystis includes some of the best-known plant pathogens in the world, responsible for a wide range of disease symptoms including stem cankers, vascular wilts and root diseases (Kile 1993). Most of these important pathogens are related to C. fimbriata Ell. & Halst. sensu lato (s.l.). Some have recently been provided with new names while others are recognized as unique based  on  phylogenetic  inference (Wingfield et al. 1996, Barnes et al. 2003, Engelbrecht & Harrington 2005,  Johnson et  al. 2005, Van Wyk et al. 2007, Rodas et al. 2008). Well known tree diseases caused by Ceratocystis spp. include oak wilt caused by C. fagacearum (Bretz) Hunt (Bretz 1952, Sinclair et al. 1987), canker stain disease of plane trees (Platanus spp.) caused by C. platani Engelbrecht et Harrington (Engelbrecht & Harrington 2005) and wattle wilt of Acacia mearnsii De Wild. caused by C. albifundus M. J. Wingf., De Beer and M. J. Morris (Morris et al. 1993, Wingfield et al. 1996). Many species,  particularly those  in  the C. coerulescens (Münch) Bakshi species complex are agents of sap stain of lumber (Münch 1907) and various species, especially in the C. moniliformis Hedgcock s.l.  species complex, appear to be saprophytes (Davidson 1935, Van Wyk et al. 2006b).
Ceratocystis spp. residing in the C. fimbriata s.l. species complex require wounds for infection (DeVay et al. 1963,  Kile 1993).   These wounds can emerge from wind and   hail damage, growth cracks, insect and other animal damage as well as human activities such as grafting and pruning. Insects carry the Ceratocystis spp., which are ecologically adapted to this mode of dissemination, to the wounds, where infection can take place (DeVay et al. 1963, Kile 1993). Stumps remaining from freshly harvested trees are also commonly infected by species of Ceratocystis (Roux et al. 2004). Recent studies have shown that artificially induced wounds are also commonly infected by Ceratocystis spp. and provide an opportunity to trap species infecting wounds from the environment  (Barnes et al. 2003, Roux et al. 2004, Rodas et al. 2008).
Relatively little is known regarding Ceratocystis spp. occurring in Africa (Roux et al. 2005). During the course of the last two decades, there have been numerous studies investigating these fungi on trees in the region. These have largely emerged from the discovery of a serious wilt disease of Acacia spp. now known to be caused by C. albifundus (Morris et al. 1993, Wingfield et al. 1996, Roux et al. 2001a,b, 2005). More recently, C. fimbriata s.l. was reported to result in rapid wilting and death of Eucalyptus spp. in the Republic of Congo (Roux et al. 2000) and Uganda (Roux et al. 2001a). This fungus has also been isolated from wounds on Eucalyptus trees in South Africa (Roux et al. 2004) but although it was pathogenic in inoculation tests, it has not been associated with disease under natural conditions.  Most recently, two other species of Ceratocystis, pirilliformis Barnes and M.  J.  Wingf.  and C.  moniliformis  have been recorded from Eucalyptus spp. in South Africa (Roux et al. 2004).
Population growth and globalisation is placing increasing pressure on native forests in Africa. For this reason, extensive forestry programmes, largely based on non-native species have been established. Diseases have already emerged as presenting serious constraints to the long term sustainability of forest plantations in Africa (Gibson 1964, Roux et al. 2005) and this is likely to be an increasing trend in the future. Concern regarding diseases has prompted surveys for important groups of tree  pathogens  including species of Ceratocystis. The aim of this study was thus to expand current knowledge relating to Ceratocystis spp. in Africa, particularly  those  occurring  on wounds on non-native A. mearnsii and Eucalyptus spp. in eastern and southern Africa.

MATERIALS AND METHODS

Collection of isolates

Isolates were collected from A. mearnsii and Eucalyptus spp. at four localities  (Piet Retief, Tzaneen, Pietermaritzburg and Lothair) in South Africa and one each in Malawi (Zomba Mountain), Tanzania (Njombe) and Kenya  (Thika).  Collections  were  made from the stumps of freshly felled Eucalyptus spp. and A. mearnsii as well as from artificially induced wounds on the stems of Eucalyptus trees. In the case of the stumps, samples were collected between four days and four weeks after felling, by removing pieces of wood displaying stained vascular tissue and/or the presence of fungal growth.
Stem wounds were made on Eucalyptus  trees using the technique previously described  by Barnes et al. (2003). Twenty trees were selected randomly at each study site and wounds were made on the stems, approximately 1.5 meters from the ground. Approximately 100 cm2 of bark was removed from the stems to expose the cambium. A horizontal slit was made into the xylem of the wound, approximately five mm deep. Samples were collected after six weeks by removing a piece of wood and bark from the top and bottom corners of the wound site and transferred to  the  laboratory in brown  paper bags for further study.
Wood sections were examined for the presence of  fruiting  structures  of  Ceratocystis spp. In addition, wood pieces displaying vascular discoloration were baited for Ceratocystis spp. by placing these between two carrot slices (five mm thick) and incubating them at 25°C for 7-10 days (Moller & DeVay 1968).  Pieces of wood were  also incubated in containers with moist tissue paper at 25°C for seven days to induce the formation of fruiting structures.
Once ascomata of Ceratocystis spp. were found, spore masses were lifted from their apices and transferred to 2% (w/v) malt extract agar (MEA) (Biolab, Midrand, South Africa) supplemented with streptomycin sulphate (0.001 g vol-1, SIGMA, Steinheim, Germany). Plates were then incubated at approximately 25ºC under natural day/night conditions. Isolates were purified on 2% MEA and are maintained in the culture  collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa. Representative isolates  were  also  deposited with the Centraalbureau voor  Schimmelcultures (CBS),  Utrecht,  The Netherlands.  In order  to prepare herbarium specimens, cultures bearing fruiting structures were dried on 30% glycerol and deposited with the National Fungal Herbarium of South Africa (PREM), Pretoria.

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Morphology and growth in culture  

All isolates collected in this study were grouped based on their  culture morphology on  2% MEA after five days and were then studied microscopically for further differentiation. Representative isolates of each group were selected for further identification using DNA sequence comparisons.
For identification based on morphology, pure cultures were  maintained on  2% MEA  until fruiting structures formed. Fungal structures were mounted on glass slides in lactic acid and examined under a Zeiss Axioskop microscope. Images were captured using a HRc Axiocam digital camera and Axiovision 3.1 software (Carl Zeiss Ltd., Germany). Fifty measurements were made for each taxonomically relevant structure  and averages and standard deviations (st. dev) were determined for each of these structures. Measurements are presented in this study as (minimum -) mean minus st. dev.  – mean plus st. dev. (- maximum). Colours of cultures were defined based on the mycological colour charts of Rayner (1970).
Two test isolates of each species were selected to study growth in  culture.  These  included one chosen to represent the holotype specimen and one of the  paratypes.  Growth rates of known species were not determined. Studies of growth in culture were performed by placing an agar disk (four mm diameter) overgrown with mycelium (mycelial side down) of selected five-day old isolates at the centres  of  90mm  Petri dishes containing 2% MEA. Petri dishes were incubated in the dark at temperatures ranging from 5°C to 35°C at 5°C intervals.  Colony  diameters  were  measured  after seven days. Two measurements, perpendicular to each other, were made  for  each  culture.  Five  replicates of each test  strain  were used at each temperature and averages  of the ten measurements taken for each isolate were computed. The entire  experiment  was repeated once.

DNA isolation, PCR reactions and sequence analyses

DNA of representative Ceratocystis isolates (Table 1) was extracted using the method described by Van Wyk et al. (2006a). Three gene regions  were  amplified  for  sequencing and phylogenetic analyses. The  ribosomal  RNA  Internal  Transcribed  Spacer regions (ITS) 1 and 2, and the 5.8S operon, were amplified using the primers ITS1 and ITS4 (White et al. 1990). Part of the beta-tubulin (β-tubulin) gene was  amplified with primers Bt1a and Bt1b (Glass & Donaldson 1995) and part of the Transcription Elongation Factor-1α (EF-1α) gene was amplified using the primers EF1F and EF1R (Jacobs et al. 2004).
Polymerase chain reaction (PCR) mixtures, for all three gene regions, consisted of 1 x Expand HF Buffer containing 1.5 mM  MgCl2  (Roche  Diagnostics,  Mannheim, Germany, supplied with the enzyme), 200 µM of each dNTP, FastStart Taq enzyme (2U) (Roche Diagnostics, Mannheim, Germany), 200 ηM of the forward and reverse primers, and 2-10 ng DNA. Reactions were adjusted to a total volume of 25 µL with sterile water. The PCR programme was set for 4 min at 95°C for initial denaturation of the DNA. This was followed by 10 cycles consisting of a denaturation step at 95°C for 40s, an annealing step for 40s at 55°C and an elongation step for 45s at 70°C. Subsequently, 30 cycles consisting of 94ºC for 20s, 55ºC for 40s with a 5s  extension  step after each cycle, and 70ºC for 45s were performed. A final step of 10 min at 72ºC completed the programme. Amplification of the DNA for the three gene regions was confirmed under ultraviolet (UV) illumination using gel  electrophoresis  with  2%  agarose in the presence of ethidium bromide. Amplicons were purified using 6% Sephadex G-50 columns following the manufacturer’s instructions  (Steinheim,  Germany).
PCR amplicons were sequenced in both directions using the ABI PRISM™ Big DYE Terminator Cycle Sequencing Ready Reaction Kit (Applied BioSystems, Foster City, California, USA), with the same primers as those used for DNA amplification.  Sequencing reactions were run on an ABI PRISM™ 3100 Autosequencer (Applied BioSystems, Foster City, California, U.S.A) and sequences were analysed  using  Sequence Navigator version 1.0.1 (Applied BioSystems, Foster City, California, USA). Sequences were compared with those of closely related Ceratocystis and Thielaviopsis spp. obtained from GenBank (http://www.ncbi.nlm.nih.gov), resulting in three datasets. The first set comprised three gene regions for isolates representing the C. fimbriata s.l. species complex, the second of the ITS gene region of Thielaviopsis spp. together with a small number of Ceratocystis spp., and the third set was made up of three gene regions  for species in the C.  moniliformis s.l.  species complex.  Sequences were aligned using  the web interface (http://align.bmr.kyushu-u.ac.jp/mafft/software/) of MAFFT (Katoh et al. 2002) and confirmed manually.

ACKNOWLEDGEMENTS 
PREFACE 
Chapter 1  BIOLOGY AND ECOLOGY OF CERATOCYSTIS SPECIES WITH AN EMPHASIS ON THEIR INSECT ASSOCIATIONS
1.1. INTRODUCTION
1.2. TAXONOMIC HISTORY OF THE GENUS CERATOCYSTIS
1.3. IMPORTANCE OF CERATOCYSTIS SPP.
1.3.1. Ceratocystis spp. infecting agricultural crops
1.3.2. Ceratocystis spp. infecting fruit-tree crops
1.3.3. Ceratocystis spp. as pathogens of forest and plantation trees
1.3.4. Staining and/or saprophytic Ceratocystis spp. on forest trees
1.4. WOUNDS AS INFECTION SIGHTS FOR CERATOCYSTIS SPP.
1.5. INSECT ASSOCIATIONS WITH CERATOCYSTIS SPP.
1.5.1. Interdependence of insects and their associated Ceratocystis spp.
1.5.2. Adaptations of Ceratocystis spp. for the purpose of insect dispersal
1.5.3 Categories of insects associated with Ceratocystis spp.
1.5.3.1 Bark Beetles
1.5.3.2. Nitidulid beetles
1.5.3.3. Generalist organisms
1.5.4. Association levels between insects and Ceratocystis spp.
1.6. CONCLUSIONS
REFERENCES
Chapter 2 SPECIES ON ACACIA MEARNSII AND EUCALYPTUS SPP. IN EASTERN AND SOUTHERN AFRICA INCLUDING SIX NEW SPECIES
ABSTRACT
2.1. INTRODUCTION
2.2. MATERIALS AND METHODS
2.2.1. Collection of isolates
2.2.2. Morphology and growth in culture
2.2.3. DNA isolation, PCR reactions and phylogenetic analyses
2.2.4. Pathogenicity tests
2.3. RESULTS
2.3.1. Collection of isolates
2.3.2. Morphology and growth in culture
2.3.3. Phylogenetic analyses Taxonomy
2.3.4. Pathogenicity tests
2.4. DISCUSSION
REFERENCES
Chapter 3  POPULATION ANALYSES OF CERATOCYSTIS ALBIFUNDUS IN SOUTHERN AND EASTERN AFRICA SUGGEST A PROGRESSIVELY SOUTHERLY HOST COLONISATION ROUTE
ABSTRACT
3.1. INTRODUCTION
3.2. MATERIALS AND METHODS
3.2.1. Isolates
3.2.2. DNA extraction, PCR amplification and allele size determination
3.2.3. Genotypic diversity
3.3. RESULTS
3.3.1. Isolates
3.3.2. Allele size determination
3.3.3. Genotypic diversity
3.4. DISCUSSION
REFERENCES
Chapter 4  INSECT ASSOCIATES OF CERATOCYSTIS ALBIFUNDUS AND PATTERNS OF ASSOCIATION IN A NATIVE SAVANNA ECOSYSTEM IN SOUTH AFRICA
ABSTRACT
4.1. INTRODUCTION
4.2. MATERIALS AND METHODS
4.2.1. Study areas
4.2.2. Traps and bait
4.2.3. Collection of insects from the “Native” study area
4.2.4. Collecting of insects from “Non-native” study area
4.2.5. Presence of fungal propagules on insect bodies
4.2.6. Isolation of fungi
4.2.7. Identification of isolates
4.2.8. Statistical analyses of data
4.3. RESULTS
4.3.1. Collection of insects from the “Native” study area
4.3.2. Collection of insects from “Non-native” study area
4.3.3. Presence of fungal propagules on insect bodies
4.3.4. Identification of isolates
4.3.5. Association of fungi with insects
4.3.6. Statistical analyses of data
4.4. DISCUSSION
REFERENCES
Chapter 5  FACTORS INFLUENCING INFECTION OF ACACIA MEARNSII BY THE WILT PATHOGEN CERATOCYSTIS ALBIFUNDUS IN SOUTH AFRICA
ABSTRACT
5.1. INTRODUCTION
5.2. MATERIALS AND METHODS
5.2.1. Preparation of inoculum
5.2.2. Wounding and inoculation of trees
5.2.3. Assessment of infection
5.2.4. Statistical analyses
5.3. RESULTS
5.3.1. Assessment of infection
5.3.2.1. Time after wounding
5.3.2.2. Pre-inoculation with O. quercus
5.3.2.3. Influence of stem diameter on infection
5.4. DISCUSSION
REFERENCES 
SUMMARY

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