Peter McMahon

Vascular Streak Dieback of cacao in Southeast Asia and Melanesia: in planta detection of the pathogen and a new taxonomy

Vascular Streak Dieback (VSD) disease of cacao (Theobroma cacao) in Southeast Asia and Melanesia is caused by a basidiomycete (Ceratobasidiales) fungus Oncobasidium theobromae (syn. =Thanatephorus theobromae). The most characteristic symptoms of the disease are green-spotted leaf chlorosis or, commonly since about 2004, necrotic blotches, followed by senescence of leaves beginning on the second or third flush behind the shoot apex, and blackening of infected xylem in the vascular traces at the leaf scars resulting from the abscission of infected leaves. Eventually the shoot apex is killed and infected branches die. In susceptible cacao the fungus may grow through the xylem down into the main stem and kill a mature cacao tree. Infections in the stem of young plants prior to the formation of the first 3-4 lateral branches usually kill the plant. Basidiospores released from corticioid basidiomata developed on leaf scars or along cracks in the main vein of infected leaves infect young leaves. The pathogen commonly infects cacao but there are rare reports from avocado. As both crops are introduced to the region, the pathogen is suspected to occur asymptomatically in native vegetation. The pathogen is readily isolated but cultures cannot be maintained. In this study, DNA was extracted from pure cultures of O. theobromae obtained from infected cacao plants sampled from Indonesia. The internal transcribed spacer region (ITS), consisting of ITS1, 5.8S ribosomal RNA and ITS2, and a portion of nuclear large subunit (LSU) were sequenced. Phylogenetic analysis of ITS sequences placed O. theobromae sister to Ceratobasidium anastomosis groups AG-A, AG-Bo, and AG-K with high posterior probability. Therefore the new combination Ceratobasidium theobromae is proposed. A PCR-based protocol was developed to detect and identify C. theobromae in plant tissue of cacao enabling early detection of the pathogen in plants. A second species of Ceratobasidium, Ceratobasidium ramicola, identified through ITS sequence analysis, was isolated from VSD-affected cacao plants in Java, and is widespread in diseased cacao collected from Indonesia.

The Role of Glutathione in the Uptake and Metabolism of Sulfur and Selenium

Sulphur (S) is essential for plant growth since it is a constituent of the two S-amino acids, cysteine and methionine, which occur in protein. S also occurs in plants in various compounds of low molecular weight, many of which are secondary compounds. They include the glucosinolates found in many Brassica species and the non-protein S-amino acids found in most Allium species. In some species these can be quantitatively important sources of S. In general, however, the abundance of S in plants is linked to the abundance of N in protein which contains both N and S. It therefore follows that one of the main determinants of the N:S ratio in plants is the frequency of the protein S-amino acids relative to the total number of protein amino acids. In vegetative plants, the N:S ratio is determined primarily by the amino acid sequence of the individual constitutive proteins and the abundance of each protein. The proteins of vegetative cereals are thought to have a N:S ratio (expressed on a molar basis) of about 30 (Djikshoorn and van Wijk 1967, Bolton et al. 1976). On the other hand, the molar N:S ratio of generative tissue depends on the frequency of the S-amino acids in the various storage proteins and the relative amounts of each protein that are made. This can be very variable as it depends on the level of N and S nutrition. Plants, unlike animals, acquire their S as sulphate from soil via an energy-requiring sulphate-specific carrier mechanism. The activity of the uptake mechanism is tightly controlled. Plants also regulate the rate at which they assimilate sulphate-S into organic S. Further, since almost all of the organic S in plants occurs in S-amino acids, then the acquisition and assimilation of inorganic S must be coordinated with the acquisition and availability of inorganic N. Thus, plants must have mechanisms for sensing the N status of the plant and using this information to adjust the rate of S acquisi-

Phosphonate applied by trunk injection controls stem canker and decreases Phytophthora pod rot (black pod) incidence in cocoa in Sulawesi.

Stem canker and Phytophthora pod rot (PPR) or black pod caused by Phytophthora palmivora are serious diseases of cocoa (Theobroma cacao L.) in Sulawesi, Indonesia, causing high yield losses for smallholders, possibly exceeded only by losses due to the cocoa pod borer (CPB), Conopomorpha cramerella. Potassium phosphonate (phosphite) applied by trunk injection has been demonstrated to effectively control canker and PPR in Papua New Guinea. The method was tested in a field trial in south-east Sulawesi. Fifty trees were injected with phosphonate, 50 with water and 50 were left untreated. Phosphonate was applied at a rate of ∼16 g active ingredient per tree per year, depending on the size of each tree. Trees were evaluated each month for canker severity, for PPR incidence and for CPB incidence and severity. From 4 months after the initial injection, trees treated with phosphonate had negligible levels of canker. Over a 2.5-year period, phosphonate significantly decreased PPR incidence. Cycles of PPR infection occurred in the wet season with PPR incidence fluctuating from less than30% to greater than 75%. These fluctuations might have been due to variations in rainfall causing natural cycles of sporulation and infection. CPB incidence did not differ significantly between treatments. Since trunk injection of phosphonate effectively controls stem canker and decreases PPR in the long term it provides a valuable option for the management of these diseases for cocoa smallholders.

On-farm selection for quality and resistance to pest/diseases of cocoa in Sulawesi: (i) performance of selections against cocoa pod borer, Conopomorpha cramerella

In Sulawesi, Indonesia, cocoa smallholdings are seriously affected by the cocoa pod borer (Conopomorpha cramerella, CPB). CPB is detrimental to both cocoa production and bean quality, and is a major concern to cocoa smallholders, processors, exporters and the international market. An Australian Centre for International Agricultural Research (ACIAR)-funded project was initiated to develop a locally applicable, farmer-participatory methodology for selecting and testing promising cocoa genotypes on farms. In a trial established on a farm in South-East Sulawesi, local Indonesian and international cocoa selections were propagated clonally by side-grafting onto mature trees of mixed genotype and evaluated for 2 years for pod value, quality and resistance to pest/diseases. Local selections were based on the observations of farmers, extension officers or researchers and included a number of clones specifically selected for resistance (or susceptibility, as checks) to CPB. Our results on the CPB-infestation of 34 clones in the trial indicate the potential of employing on-farm selection and testing to improve cocoa farms. Severity of infestation was determined in ripe pods according to whether the proportion of beans damaged by CPB larvae was light (less than 10%), moderate (10–50%) or severe (over 50%). Total CPB incidence in ripe pods for most of the clones was high, exceeding 75%, and was correlated with severe and light incidence, both of which varied more. The data support a model that predicts severe infestation inflicting bean losses occurs above a critical threshold of total CPB incidence. The cumulative CPB incidence in infested pods was significantly lower in a local selection, Aryadi 2, which also had fewer larval entry holes and a low exit/entry ratio indicating a degree of resistance to CPB. The incidence of lightly infested pods was significantly lower in the susceptible controls. Pod hardness was moderately high in Aryadi 2. However, VSD3 (a local selection), Scavina12, KKM22 and BR25, had low levels of severe infestation and/or high incidences of light infestation, but had relatively soft pods. This suggests that factors in addition to pod hardness might be involved in resistance, a possibility also supported by the positive correlation of CPB incidence in ripe pods with that in immature pods and the larger seasonal fluctuations of CPB incidence observed in resistant clones, compared to susceptible clones. In a mixed genotype stand, the lower CPB incidence observed in some clones might be explained partly by pest non-preference.

On-farm selection for quality and resistance to pest/diseases of cocoa in Sulawesi: (II) Quality and performance of selections against Phytophthora pod rot and vascular-streak dieback.

The cocoa industry in Sulawesi, the main region of cocoa production in Indonesia, is threatened by destructive diseases, including vascular-streak dieback (VSD) caused by the basidiomycete Oncobasidium theobromae and stem canker and Phytophthora pod rot (PPR) or black pod, caused by Phytophthora palmivora. Using the considerable genetic diversity of cocoa on farms, host resistance was identified and tested with the participation of farmers. Forty-nine local and international cocoa selections with promising resistance characteristics (as well as susceptible controls) were side-grafted onto mature cocoa in a replicated trial with single-tree plots. Developing grafts were assessed in the dry season for severity of VSD infection, scored from 0 (no infection) to 4 (graft death). All of the 49 clones in the trial became infected with VSD in at least some replicates. Average severity varied from 0.2 to 1.6. Potential VSD-resistance was found in eight clones, including DRC 15, KA2 106 and a local Sulawesi selection, VSD2Ldg. Some of the most susceptible clones were local Sulawesi selections from areas with a history of little or no VSD. Thirty-four pod-bearing clones were evaluated over a 2-year period for yield, quality and resistance to natural infections of PPR. Cumulative PPR incidence for all clones was 22% but varied from 8.6 to 43% among clones. Clones with less than 15% PPR incidence were designated as resistant, including DRC 16 and local Sulawesi selections, Aryadi 1, Aryadi 3 and VSD1Ldg. Scavina 12 was moderately resistant in the trial with a PPR incidence of 23%. Cumulative incidences of the mirid, Helopeltis spp., determined in the same evaluation period, indicated that DRC16 was the most susceptible clone with an incidence of 52% in ripe pods and 23% in immature pods. In comparison, KKM4 showed evidence of resistance to Helopeltis spp., with incidences of 34 and 0.8% in ripe and immature pods, respectively. The impact of diseases and pests (including cocoa pod borer) on bean losses and bean quality varied between clones but generally the bean size (or bean count) was affected more than the fat content or shell content.

Vascular Streak Dieback (Ceratobasidium theobromae): History and Biology

Vascular streak dieback (VSD) caused by the basidiomycete, Ceratobasidium theobromae (syn. Oncobasidium theobromae, Thanatephorus theobromae), is one of the most important diseases of cacao in the Southeast Asian/Melanesian region, causing branch dieback with infections capable of killing seedlings and mature trees of susceptible cacao varieties. The only known host is Theobroma cacao, but as a new encounter disease it is apparent that the fungus transferred to cacao from an original host, endemic to the region, which so far remains unidentified. Basidiospores that initiate infection are short-lived and dispersed by wind only for short distances. VSD is patchy in distribution but can be severe locally and, recently in Indonesia, has influenced farmers to convert to crops other than cacao. In Malaysia this disease, among other factors, influenced growers to replace cacao with oil palm. The causal pathogen and disease symptoms were first described in Papua New Guinea following a severe epidemic in the 1960s: a unique wind-dispersed basidiomycete pathogen was identified that infected young leaves of cacao seedlings and mature trees, colonizing the xylem and resulting, after about 3 months, in characteristic symptoms of leaf chlorosis, leaf fall, and branch dieback or seedling mortality. Sporulation was observed to occur on the monilioid hyphae emerging from leaf scars. Quite recently, within the last decade, symptoms of leaf marginal and tip necrosis, associated with a longer period of attachment before leaf abscission, have become dominant, replacing the formerly characteristic chlorotic symptoms. As a consequence of delayed abscission, sporulation now frequently occurs on hyphae emerging through cracks in the petiole or leaf mid-rib. The pathogen associated with the newer symptoms, C. theobromae, appears morphologically and genetically identical to the species previously described. However, haplotypes based on ITS sequences have been identified within the region, indicating that some regional genetic variability occurs. Just as in VSD-infected cacao displaying chlorotic symptoms, infections associated with the more recent (necrotic) symptoms spread from initially infected leaves via the xylem to neighboring leaves and, in more susceptible genotypes, reach the tip causing dieback. The two sets of symptoms (chlorotic and necrotic) may occur in the same area and even on the same tree, and their relative frequency is influenced by the season. At higher altitudes and in some genotypes the former symptoms of leaf chlorosis are predominant. Disease severity also decreases with altitude. It remains uncertain whether the recent increase in VSD severity in Sulawesi, East Java, and other areas in the region is linked to the new symptoms. Since the incubation period for the disease is long (3 months or more) it is important to keep seedlings in quarantine for about 6 months before distribution for planting in other locations. As C. theobromae is a vascular pathogen, control by fungicides is difficult, and although effective systemic triazole fungicides have been identified, they are generally too costly for smallholder farmers. The disease is best controlled by sanitation pruning and the adoption of resistant genotypes. Further work is under way to combine resistance with other characteristics such as superior cacao bean quality and yield.

EFFECT OF ORGANIC AND INORGANIC AMENDMENTS ON PRODUCTIVITY OF COCOA ON A MARGINAL SOIL IN SULAWESI, INDONESIA

Reduced soil fertility and damage from pests and diseases have contributed to a decline in productivity of cocoa (Theobroma cacao L.) smallholdings in Sulawesi, Indonesia over the last decade. In a trial on a marginal, acidic soil in South Sulawesi, young PBC123 cocoa trees were supplied with compost, mineral fertiliser (NPK fertiliser and urea) or dolomite, alone and in combination. After 20 months, the trees supplied with compost were taller, flowered more profusely and had a five-fold higher dry bean yield than other treatments. Treatments had no impact on incidence of cocoa pod borer, Phytophthora pod rot and vascular streak dieback. All of the trees supplied with compost survived, while the control, mineral-fertiliser- and dolomite-treated trees had a mortality rate of 22–45% and symptoms of interveinal necrosis. Leaf concentrations of N, P and K were within the normal range in all treatments. In the control and mineral fertiliser treatments, leaf concentrations of Ca (0.28–0.30%) and Mg (0.11–0.15%) were deficient, but were higher in trees supplied with compost (0.78–1.21% and 0.26–0.29%, respectively). The Mg/K ratio in soil-exchangeable cations and leaves was increased three-fold by the combined compost/dolomite treatment. Supplying mineral fertiliser alone resulted in 3.3 cmol kg−1 exchangeable Al, compared to 2.2 cmol kg−1 in control soils. Since 10 kg tree−1 year−1 compost was supplied, a rate that is not practical on most cocoa smallholder farms, further investigation of cost-effective applications of organic matter in conjunction with appropriate formulations of inorganic fertilisers is recommended.