17th INTERNATIONAL COCOA RESEARCH CONFERENCE
HILTON HOTEL Yaoundé - CAMEROON 15 - 20 OCTOBER 2012
Molecular identification of Armillaria isolates associated with root-rot disease of cacao in Cameroon J. Roux, M. Mbenoun, A.C. Misse, M.P.A. Coetzee and M.J. Wingfield DST/NRF Centre of Excellence in Tree Health Biotechnology (CTHB), Department of Microbiology and Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), Private Bag X20, University of Pretoria, Hatfield, 0028, South Africa Armillaria root-rot is one of the most serious root diseases of perennial tree crops in tropical Africa. The disease was first reported on cacao in Ghana during 1927. It was initially referred to as ‘collar crack’ due to typical longitudinal cracks that are formed in the bark at the tree collar, as a result of mechanical pressure from fungal fans colonizing the cambium. These fungal fans are typical of species in the genus Armillaria. The pathogen associated with the disease was identified as A. mellea (Valh Ex. Fr.). However, A. mellea is a taxonomic complex that currently includes a number of morphologically indistinguishable species, the true identity of the species causing root-rot on cacao in Africa is thus uncertain. More recent studies have shown that DNA sequence comparisons can effectively differentiate among Armillaria species from Africa with uniform morphologies. In this study we applied a molecular approach to characterize Armillaria isolates associated with root-rot disease of cacao in Cameroon. Isolates were collected in three cacao plantations and the ITS and IGS nuclear DNA regions were sequenced and used in phylogenetic analyses. Phylogenetic trees placed the isolates from Cameroon in a group that included sequences representing the A. fuscipes sensu lato clade. Further work, however, is needed to elucidate the taxonomy of A. fuscipes s.l. as it contains sub-clades that possibly represent separate species.
Molecular identification of Armillaria isolates associated with root-rot disease of cacao in Cameroon Jolanda Roux, Michael Mbenoun (Department of Microbiology & Plant Pathology, Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria (UP), Pretoria, 0028; [email protected]
), Alain C. Misse (Department of Microbiology & Plant Pathology, FABI, South Africa and IRAD, Yaounde, Cameroon), Martin P. A. Coetzee (Department of Genetics, FABI, UP), Didier Aime Begoude (IRAD, Yaounde, Cameroon) & Michael J. Wingfield (Department of Microbiology and Plant Pathology, FABI, UP) http://fabinet.up.ac.za & [email protected]
INTRODUCTION Armillaria root-rot is one of the most widespread and important diseases of plants globally. In tropical Africa, it causes serious damage on many perennial tree crops, often resulting in tree death. The first report of the disease on Theobromae cacao in Africa originated from Ghana in 1927. It was initially referred to as ‘collar crack’ due to typical longitudinal cracks that are formed in the bark at the tree collar, as a result of mechanical pressure from mycelial fans colonizing the cambium. These fans are typical of species in the genus Armillaria. The taxonomy of Armillaria has undergone significant revision in recent years, particularly due to the application of DNA sequence comparisons and phylogenetic inference. Many reports of Armillaria diseases in Africa, prior to the advent of these new methods, referred to the causal agent as A. mellea (Valh Ex. Fr.) or A. heimii. However, these reports were based only on morphology and microscopic features of the basidiocarps, and in a few cases mating tests. These have since been shown to be unreliable in differentiating species of Armillaria. Based on DNA sequence data and other molecular characters, it is now known that A. mellea is a taxonomic complex that includes numerous morphologically indistinguishable species. Similarly, A. heimii has also been shown to represent a species complex that includes at least two taxa, i.e. A. fuscipes and an undescribed species (Coetzee et al. 2005). Armillaria heimii and A. fuscipes have been shown to be synonyms, with the name A. fuscipes having priority. Armillaria species reported from Cameroon include A. camerunensis (Henn.) Volk & Burdsall, A. elegans and A. heimii (A. fuscipes). These reports are, however, mostly based on morphological features and no isolates from cacao in the country have been identified using DNA sequence studies. The true identity of the species causing root-rot on cacao in Africa is, thus, uncertain.
• Typical symptoms of Armillaria root rot were observed on T. cacao in three plantations in Cameroon (Figure 1A-E). These plantations were situated in the South West and Centre regions of the country. • Affected trees ranged from 5 years in age to more than 10 years. • Typical Armillaria cultures, showing abundant rhizomorph growth (Figure 1F) were obtained from symptomatic material. • Sequence data for the ITS and IGS-1 regions were successfully obtained for all isolates. • All isolates from T. cacao in Cameroon grouped into a single phylogenetic clade, most closely related to A. fuscipes. Considerable sub-structure was observed in the phylogenetic clade in which Cameroonian isolates grouped. Cacao isolates from Cameroon fell into three of these sub-clades and grouped according to their geographic origin in the country.
AIM Determine the cause of Armillaria root rot of T. cacao in Cameroon using DNA sequence comparisons and phylogenetic methods.
MATERIALS & METHODS • Samples of infected roots and stems showing white mycelial mats typical of Armillaria species were obtained from T. cacao trees in the Bokito, Ngat and Kumba areas of Cameroon. Care was taken not to expose the white mycelial mats completely, so as to avoid contamination of the material during transport to the laboratory. • In the laboratory, the bark of infected material was separated from the wood to expose the mycelial mats, whereafter small pieces of mycelium were transferred aseptically to a selective medium (Harrington et al. 1992). • Cultures, showing typical growth of Armillaria species were selected for further study. • Small pieces from the rhizomorph tips of each culture were transferred and stored on malt yeast agar medium (MYA). • DNA was extracted from cultures representing different areas in Cameroon using the technique described by Möller et al. (1992). • The IGS-1 (inter-genic spacer 1) and ITS (internally transcribed spacer 1, 2 including the 5.8S gene) regions of the rDNA operon were amplified and sequenced using published protocols (Coetzee et al. 2005). • DNA sequences were aligned using the online interface of MAFFT (http://mafft.cbrc.jp/alignment/server/) and compared with sequences on GenBank. • Phylogenetic trees were generated based on parsimony (using PAUP*) and maximum likelihood (using PHYML).
ACKNOWLEDGMENTS We thank the Department of Science and Technology (DST) and National Research Foundation (NRF) in South Africa for funding. We are also grateful to farmers, conservation agents and all concerned members of the public in Cameroon who reported tree decline and assisted the research team with field studies.
Figure 1: Symptoms and signs of Armillaria root rot of Theobromae cacao. (A) Dead T. cacao trees, often in clumps due to root to root spread of the pathogen, (B) basal canker characterized by cracking and discolouration of the bark, (C) leading edge of basal canker, with white mycelial mats of Armillaria sp. visible in the cracks, (D) internal symptoms of Armillaria infection showing cracking and white mycelium in the cracks, (E) mycelial mats in the xylem of recently dead tree, (F) Armillaria sp. culture on MEA, showing typical rhizomorph growth.
CMW34124 Theobroma cacao Cameroon Bokito CMW34126 Theobroma cacao Cameroon Bokito CMW34128 Theobroma cacao Cameroon Bokito CMW3173 Tectona grandis Zambia CMW10115 Acacia albida Zimbabwe CMW10116 Newtonia buchananii Zimbabwe CMW36818 Theobroma cacao Cameroon Ngat CMW4455 Camellia sinensis Zimbabwe CMW4456 Brachystegia utilis Zimbabwe 90 CMW11653 Citrus sinensis Zimbabwe CMW11649 Citrus sinensis Zimbabwe 100 CMW11662 Pinus Zimbabwe CMW11650 Pinus Zimbabwe 99 CMW1 Prunus sp. Zimbabwe CMW3152 Cameroon CMW36821 Theobroma cacao Cameroon Kumba CMW36823 Theobroma cacao Cameroon Kumba 99 CMW36825Theobroma cacao Cameroon Kumba CMW36824 Theobroma cacao Cameroon Kumba CMW2717 Pinus elliottii South Africa CMW2740 Pinus patula South Africa CMW3164 Pelargonium asperum La Reunion CMW4871 Widdringtonia whytei Malawi CMW4873 Indigenous shrub Malawi 100 CMW4949 Pinus elliottii Tanzania CMW4950 Pinus strobes Tanzania CMW4953 Pelargonium asperum La reunion CMW10165 Prunus persica Zimbabwe A. fuscipes CMW4874 Araucaria cunninghamii Zimbabwe CMW9963 Prunus persica Zimbabwe CMW7184 Cypress sp Kenya CMW7187 Camellia sinensis Kenya CMW15580 Camellia sinensis Kenya CMW15582 Camellia sinensis Kenya CMW8971 Pinus patula Ethiopia CMW5846 CMW5846 Pinus patula_Ethiopia CMW5837 Pinus patula Ethiopia CMW8967 Cordia alliodora Ethiopia CMW8969 Acacia abyssinica Ethiopia CMW5844 Pinus patula_Ethiopia
Figure 2: Phylogenetic tree of IGS-1 gene region showing position of cacao isolates from Cameroon, relative to their closest phylogenetic neighbor, A. fuscipes. Isolates sequenced in this study are in red. Bootstrap values are indicated above the branches.
This is the first formal report of Armillaria root rot of T. cacao in Cameroon. The disease was characterized by typical yellowing of the crowns of infected trees, wilting and death of these trees. Closer inspection showed the presence of cracks and discolouration at the bases of trees and the presence of white mycelial mats when the cambium and wood of trees were exposed. Typical of Armillaria root rot, dead trees often occurred in patches in the plantations. Isolates from T. cacao in Cameroon grouped in a phylogenetic clade which has previously been suggested to represent a distinct and undescribed species of Armillaria (Coetzee et al. 2005). Isolates in this clade had previously been treated as A. heimii, which is now known as Clade B and sister to A. fuscipes sensu stricto. This species includes a previously collected isolate of Armillaria from Cameroon, from an unknown host. This clade is sister to A. fuscipes sensu stricto. Sub-structuring within this clade may suggest the presence of multiple species in Cameroon, however, additional studies are needed to confirm this. The relationship of Armillaria species from T. cacao in Cameroon, to A. camerunensis and A. elegans, previously reported from the country, is not possible to determine. There are no living cultures for A. camerunensis or A. elegans from Cameroon. Furthermore, no basidiocarps have been obtained for the fungus from cacao in the country. The management of Armillaria root rot in plantations/orchards is complex. Outbreaks on agricultural crops, including T. cacao, are often associated with shade trees and areas where native forest has been cleared to establish farms. These trees often serve as sources of inoculum, but once established, infection centres can persist on the roots and stumps of dead cacao trees for long periods of time. The containment of infection centres using trenching to prevent root to root contact between plants, and lime application has had limited success and is labour intensive.
CONCLUSIONS • Armillaria root rot of T. cacao is common in some regions of Cameroon, resulting in considerable losses to farmers. • Further work is needed to elucidate the taxonomy of A. fuscipes s.l. as it contains sub-clades that most likely represent separate species.
LITERATURE CITED • Coetzee, M.P.A., Wingfield, B.D., Bloomer, P. and Wingfield, M.J. (2005). Phylogenetic analyses of DNA sequences reveal species partitions amongst isolates of Armillaria from Africa. Mycological Research 109: 1223-1234. • Harrington TC, Worrall JJ, Baker FA. 1992. Armillaria. In: Singleton LL, Mihail JD, Rush C, editors. Methods for research on soil-borne phytopathogenic fungi. St. Paul, MN.: American Phytopathological Society Press. pp. 81-85. • Möller EM, Bahnweg G, Sandermann H, Geiger HH. 1992. A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucleic acids research 20: 6115–6116.