Sudden death of cocoa in Papua New Guinea associated with Phytophthora palmivora cankers invaded by bark beetles

An extensive survey in 1976-82 indicated that sudden death of cocoa was widespread in Papua New Guinea with losses up to 1% per month in trees over 10-yr-old, especially in neglected plantations. Over 95% trees affected had large Phytophthora palmivora canker lesions and over 90% of the cankers were invaded by bark beetles. In an intensive single year survey of one plot 18-8% of 69 healthy trees died. Ambrosiella spp., P. palmivora and Fusarium solani were isolated from wood surrounding beetle tunnels but only the last two caused extensive lesions when inoculated into the xylem of healthy trees. The evidence obtained indicated that the sudden death syndrome is initiated by P. palmivora cankers on trunks and main branches but bark beetles are a major contributory factor and fungi associated with their tunnels may contribute to the problem. There is a risk of further outbreaks of the disease as cocoa cultivars susceptible to P. palmivora continue to be planted.     

Association of Phytophthora palmivora bark canker and insect damage in cocoa in Papua New Guinea

Bark canker caused by the fungus Phytophthora palmivora has caused the death of many cocoa trees over 15 years old in Papua New Guinea in recent years. Cankers are significantly associated with channels made by the larvae of bark-damaging insects, which provide the major entry point for the pathogen. The combination of canker and insect damage may limit the economic life of cocoa to less than 15 years in affected areas.     

Cocoa-based media for culturing Phytophthora palmivora (Butl.) Butl., causal agent of black pod disease of cocoa

Green cocoa pod husk agar (GCPA), ripe cocoa pod husk agar (RCPA), green cocoabean agar (GCBA), ripe cocoa bean agar (RCBA), green cocoa mucilage agar (GCMA)and ripe cocoa mucilage agar (RCMA) were prepared and assessd for their clarity andfor potential to support mycelial growth and sporulation of P. palmivora. Oatmeal agar (OMA), potato-dextrose agar (PDA), vegetable 8 juice agar (V8JA) and pineapple crown agar (PCA) were included for comparison. The highest radial growth rates of 8.3 and 7.2 mm/day were recorded, respectively, on OMA and GCPA but these were not significantly different (P ≤ 0 05) from each other. The two media also supported good aerial mycelial growth but were not clear. Radial mycelial growth rates of 6.5, 7.0 and 6.6 mm/day were obtained on GCMA, RCPA and V8JA, respectively, and these rates were also not significantly different from each other. Of the three media, only the GCMA was clear and supported the best aerial mycelial growth. In comparison, the RCMA supported a significantly lower radial growth (4.6 mm/day) of P. palmivora than the three media. Growth rates were least on RCBA, PCA and PDA but sporulation was poorest on PDA, PCA and V8JA. GCMA was found to be the best medium based on all the growth parameters and media characteristics. GCMA has been used effectively to isolate/detect P. palmivora from infected cocoa pod tissues. Apart from differences in radial growth rate, both the GCMA and RCMA were similar in all other respects and are recommended for culturing P. palmivora. This revised version was published online in June 2006 with corrections to the Cover Date.     

Potential for biological control of tent building species of ants associated with Phytophthora palmivora pod rot of cocoa in Papua New Guinea

The tents of different ant species collected from young cocoa trees in their first or second year of bearing and free from visible pod rot, were tested for the presence of Phytopthtora palmivora by inoculating wounded cocoa pods with tent material. Tents of all species harboured viable P. palmivora but those consisting mainly of soil were more frequently positive than plant debris-type tents. Although Anoplolepis longipes, a dominant, ground nesting, non-tent building species sometimes transported inocula in the laboratory, it did not significantly increase black pod infection in the field. Trees infested with the dominant debris tent building species, Technomyrmex albipes, however, had significantly more black pod than those infested with A. longipes or trees without ants. A. longipes forms dense populations and can exclude other dominant ants and some cocoa pests; its introduction may be a potentially economical method of reducing the transmission of P. palmivora in redeveloped cocoa in Papua New Guinea.     

Phytophthora spp. from rubbertree plantations in Yunnan Province of China

Phytophthora spp. associated with leaf fall, stem canker and black stripe of rubbertree (Hevea brasiliensis Muell.-Arg.) in Xishuangbanna of Yunnan Province were isolated from leaves, bark, fruit and soil in the plantations. Of a total of 50 isolates, 42 were designated as P. citrophthora (R. E. Sm. & E. H. Sm.) Leonian, while the others were identified as P. palmivora (Butl.) Butl., P. colocasiae Rac. and P. cactorum (Leb. & Conn) Schroet. This appears to be the first report of P. citrophthora on Hevea brasiliensis.     

An in vitro investigation of Malaysian Phytophthora palmivora isolates and pathogenicity study on oil palm

Bud rot disease affecting oil palm in South American countries is reported to be caused by Phytophthora palmivora. P. palmivora is a local pathogen affecting various crops in Malaysia, and this finding caused an alarm, which prompted an investigation of pathogenicity using Malaysian P. palmivora to assess the potentials of this Oomycete to infect oil palm in Malaysia. A total of 11 P. palmivora isolates were obtained from cocoa and durian for the study. Leaf bioassays via artificial inoculation using 50,000 zoospores/ml and mycelial agar disc showed severe necrotic lesions on the infection spot of oil palm (DxP) spear leaves. Mild infection was observed in oil palm spear leaves of OxG hybrids indicating lower susceptibility against P. palmivora infection. Phylogenetic analysis using internal transcribed spacer (ITS) data revealed that Malaysian isolates were genetically similar to Colombian isolates supported by significant bootstrap values. The leaf bioassay results revealed that Malaysian oil palm materials are susceptible towards local P. palmivora infection. The Colombian P. palmivora isolates causing bud rot incidence may have evolved over a long period of time, undergone sequential genetic shift to become more virulent towards Colombian oil palm planting materials.     

Creation of BAC genomic resources for cocoa (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) for physical mapping of RGA containing BAC clones

We have constructed and validated the first cocoa (Theobroma cacao L.) BAC library, with the aim of developing molecular resources to study the structure and evolution of the genome of this perennial crop. This library contains 36,864 clones with an average insert size of 120 kb, representing approximately ten haploid genome equivalents. It was constructed from the genotype Scavina-6 (Sca-6), a Forastero clone highly resistant to cocoa pathogens and a parent of existing mapping populations. Validation of the BAC library was carried out with a set of 13 genetically-anchored single copy and one duplicated markers. An average of nine BAC clones per probe was identified, giving an initial experimental estimation of the genome coverage represented in the library. Screening of the library with a set of resistance gene analogues (RGAs), previously mapped in cocoa and co-localizing with QTL for resistance to Phytophthora traits, confirmed at the physical level the tight clustering of RGAs in the cocoa genome and provided the first insights into the relationships between genetic and physical distances in the cocoa genome. This library represents an available BAC resource for structural genomic studies or map-based cloning of genes corresponding to important QTLs for agronomic traits such as resistance genes to major cocoa pathogens like Phytophthora spp (palmivora and megakarya), Crinipellis perniciosa and Moniliophthora roreri.Communicated by J.W. Snape     

Additive inheritance of resistance to pod rot caused by Phytophthora palmivora in cocoa

Summary Quantitative inheritance of resistance to Phytophthora pod rot (Ppr) was studied in cocoa hybrid progeny from 12 Trinitario x Amazonian crosses and their reciprocal crosses. The crossing scheme was similar to a factorial design. Disease was assessed by the number and percentage of infected pods on each tree. Highly significant differences due to general combining abilities (GCA) were obtained for all characters, except for the GCA of Trinitario on total pod production. Differences for specific combining ability (SCA) were not significant for all characters. There were no significant differences between reciprocal crosses. The Trinitario clone K82 provided the only source for the hybrid progenies of strong Ppr resistance to the hybrid progenies, while K20 provided moderate resistance. Other parental clones — KA2-101, KA5-201, KEE 2, KEE 5, and KEE 52 — produced progenies which were susceptible to Ppr. It is evident that resistance to Ppr in cocoa is inherited additively. Maternal and cytoplasmic effects were assumed to have no influence on inheritance of resistance. It is also concluded that resistance to Ppr of the kind shown by K82 is likely to be horizontal resistance. Breeding for high-yielding cultivars combined with Ppr resistance is the most effective way of controlling Ppr of cocoa on the crops of growers with small holdings in Papua New Guinea.     

A small-scale screening technique for evaluating fungicides against Phytophthora palmivora pod rot of cocoa

Plots of clonal Trinitario cocoa were sprayed with fungicides in the field. Ripe pods were harvested from the trial plots and artificially inoculated with Phytophthora palmivora using various techniques, the most suitable of which proved to be the zoospore spot method. Percentage infection results from the spot inoculation tests agreed well with the field percentage infection results and it was concluded that field spraying followed by zoospore spot inoculation of detached pods is a suitable preliminary screening technique for fungicides. Three systemic fungicides metalaxyl, aluminium tris (ethyl phosphonate) and DPX-3217 in a mixture with captafol and copper oxychloride were compared with the protectant cuprous oxide. Metalaxyl was found to be the most effective of the systemic fungicides and when applied at the rate of 0·44 g ai/ha every 8 wk it gave a similar reduction in percentage infection to 4 weekly applications of cuprous oxide at the rate of 4·4 kg Cu⁺⁺/ha during the wetter part of the year when pod rot is most severe.     

Biosynthesis of scopoletin in Hevea brasiliensis leaves inoculated with Phytophthora palmivora

Inoculation of resistant (R) and susceptible (S) Hevea brasiliensisleaves with Phytophthora palmivorainduced foliar necrosis and biosynthesis of scopoletin (Scp), considered as a Heveaphytoalexin. The degree of resistance of four clones, as classified by the necrotic lesions, was related to the rapidity and intensity of Scp production. The resistant BPM-24, and marked partially resistant clone PB-235, displayed an early secretion of scopoletin that intensified and lasted longer than the weak partially resistant RRIT251, and susceptible clone RRIM600. The lesion size and amount of Scp after infection were positively correlated to the concentration of spores applied to Hevealeaves. In addition, in leaflets inoculated with high spore concentration, Scp reached the highest level earlier than those with low spore concentration. A fungitoxic effect of Scp on mycelium growth was shown in bioassays; the I₅₀ value tested on Phytophthora palmivora was relatively the same as on Phytophthora botryosa, but much lower than those found on other leaf pathogens of rubber tree, Corynespora cassiicolaand Colletotrichum gloeosporioides. The lesion size and level of Scp upon spore inoculation may be appropriate for classifying Heveaclones according to their R/S with respect to P. palmivora     

Phytophthora palmivora from Sulawesi and Java Islands,Indonesia, reveals high genotypic diversity and lack of population structure

Phytophthora palmivora is the causal agent of cocoa black pod disease, one of the primary diseases of cocoa in Indonesia. A better understanding of P. palmivora population genetics is needed to aid the development of relevant disease management strategies. This study is the first population genetic study of P. palmivora in Indonesia using microsatellite markers based on the alleles genotyping method. The microsatellite markers were used to determine the genotype of 44 P. palmivora isolates from Sulawesi (24) and Java (20) islands. The total number of observed multilocus genotypes (MLG) from both populations was 34. The genotypic diversity of P. palmivora from Sulawesi (2.90; 16.0; 0.938) and Java (2.76; 14.3; 0.930) islands was high as seen from Shannon's diversity index (H), Stoddart and Taylor's Index (G), and Simpson's Index (λ) respectively. Evenness and Nei's unbiased gene diversity exhibited similarly high levels from both populations. The linkage disequilibrium test indicated that sexual recombination occurred in the Java population (P = 0.312). Analysis of molecular variance (AMOVA) and Bayesian clustering revealed five genetic clusters, and isolates from both islands were evenly distributed across the five gene clusters. All genetic diversity was from within individuals. P. palmivora from Sulawesi and Java showed a high genotypic diversity but a lack of genetic differentiation among populations (Fst = 0.006). Both populations formed one highly diverse group. Minimum spanning network analysis showed no particular grouping of MLGs, and shared MLGs from both populations indicated long-distance migration of P. palmivora facilitated by human activities.     

Photoinhibition and Recovery of Photosynthesis in Canker-susceptible and Resistant Needles of Cypress (Cupressus sempervirens L.)

The aim of the present investigation was to test the hypothesis that the cypress canker caused by a fungus (Seiridium cardinale) infection induced effects on photosynthesis which could be related to photoinhibition and the process of recovery in susceptible and resistant needles. Photoinhibition of photosynthesis and recovery was studied in canker‐infected susceptible and resistant needles of cypress (Cupressus sempervirens L.) under controlled conditions (irradiation of detached needles to approximately 1900 μmol/m²/s). The degree of photoinhibition was determined by means of the ratio of variable to maximum chlorophyll (Chl) fluorescence (F_v/F_m) and electron transport measurements. The potential efficiency of photosystem (PS) II, F_v/F_m declined, and F_o increased significantly in canker‐susceptible needles, while F_o did not change in resistant needles. In isolated thylakoids, high light (HL) decreased the rate of whole chain and PS II activity markedly more in susceptible than in resistant needles. A smaller reduction of PS I activity was noticed only in susceptible needles. Upon subsequent dark incubation, fast recovery was noticed in both needle types and reached maximum rates of PS II efficiency similar to those noticed in non‐photoinhibited needles. The artificial exogenous electron donors such as diphenyl carbazide (DPC), NH₂OH and Mn²⁺ failed to restore the HL induced loss of PS II activity in susceptible needles, while DPC and NH₂OH significantly restored it in resistant needles. The results suggest that HL inactivates the donor side of PS II in resistant and the acceptor side of PS II in susceptible needles. The results on the quantification of the PS II reaction centre protein D1 and 33 kDa protein of water‐splitting complex following HL exposure showed pronounced differences between susceptible and resistant needles. The marked loss of PS II activity in HL‐irradiated needles was due to the marked loss of D1 protein in susceptible and 33 kDa protein in resistant needles, respectively.     

Inducible Proteins in Citrus Rootstocks with Different Tolerance Towards the Root Rot Pathogen Phytophthora palmivora

Activities of defence‐related proteins (β‐1,3‐glucanases, chitinases and peroxidases) and concentrations of total soluble phenolics were measured in roots and leaves of non‐infected and infected plants to investigate the response of different citrus rootstock genotypes to the root rot pathogen Phytophthora palmivora Butler. Infection with the pathogen increased concentrations of total proteins, total phenolics and β‐1,3‐glucanase activity in roots of all genotypes, and increases were associated with the extent of root mass reductions and thus susceptibility of the plants. Root chitinase and root peroxidase levels were slightly reduced or unaltered upon infection. β‐1,3‐Glucanase activity was also elevated in leaves of infected plants, but increases did not differ between tolerant and susceptible rootstocks. Effects of root infection on leaves were typically the reverse of effects on roots for chitinase‐ and peroxidase levels and more pronounced in susceptible rootstock genotypes. Although differences in enzyme expression were observed between susceptible and tolerant citrus seedlings, effects were usually associated with disease progression, and not with resistance to P. palmivora. It is suggested that increased activities of the proteins and soluble phenolics studied are not implicated in the primary defence to Phytophthora root diseases, but may contribute to the inhibition of the pathogen during infection in tolerant citrus.     

Modification of the PthA4 effector binding elements in Type I CsLOB1 promoter using Cas9/sgRNA to produce transgenic Duncan grapefruit alleviating XccΔpthA4:dCsLOB1.3 infection

Citrus canker caused by Xanthomonas citri subspecies citri (Xcc) is a severe disease for most commercial citrus cultivars and responsible for significant economic losses worldwide. Generating canker‐resistant citrus varieties will provide an efficient and sustainable solution to control citrus canker. Here, we report our progress in generating canker‐resistant grapefruit by modifying the PthA4 effector binding elements (EBEs) in the CsLOB1 Promoter (EBE_PthA4‐CsLOBP) of the CsLOB1 (Citrus sinensis Lateral Organ Boundaries) gene. CsLOB1 is a susceptibility gene for citrus canker and is induced by the pathogenicity factor PthA4, which binds to the EBE_PthA4‐CsLOBP to induce CsLOB1 gene expression. There are two alleles, Type I and Type II, of CsLOB1 in Duncan grapefruit. Here, a binary vector was designed to disrupt the PthA4 EBEs in Type I CsLOB1 Promoter (TI CsLOBP) via epicotyl transformation of Duncan grapefruit. Four transgenic Duncan plants with targeted modification of EBE_PthA4‐T1 CsLOBP were successfully created. As for Type I CsLOB1 promoter, the mutation rate was 15.63% (#D13), 14.29% (#D17), 54.54% (#D18) and 81.25% (#D22). In the presence of wild‐type Xcc, transgenic Duncan grapefruit developed canker symptoms similarly as wild type. An artificially designed dTALE dCsLOB1.3, which specifically recognizes Type I CsLOBP, but not the mutated Type I CsLOBP or Type II CsLOBP, was developed to infect Duncan transformants. Consequently, #D18 had weakened canker symptoms and #D22 had no visible canker symptoms in the presence of XccΔpthA4:dCsLOB1.3. Our data suggest that activation of a single allele of susceptibility gene CsLOB1 by PthA4 is sufficient to induce citrus canker disease, and mutation in the promoters of both alleles of CsLOB1 is probably required to generate citrus canker‐resistant plants. This work lays the groundwork to generate canker‐resistant citrus varieties via Cas9/sgRNA in the future.     

Role of associative bacterial interaction in the induction of tuber rot incidence in cassava (Manihot esculenta Crantz)

Cassava (Manihot esculenta Crantz), one of the leading staples in the world, has considerable scope for integration into emerging markets through efficient and environmentally sound production of a diversified range of high quality, competitive products for food, feed and industry. One of the major reasons attributed to the low productivity of cassava in India is the incidence of tuber rot in cassava incited by Phytophthora palmivora. Usually, an array of microorganisms is involved in rotting of cassava tubers in the field and in postharvest conditions. Unfortunately, the disease management practices focus mainly on fungal pathogens. In this article, we tried to investigate the role of associative bacterial pathogens in induction of tuber rot of cassava, the potential of the bacteria with P. palmivora in causing disease incidence, spread and rotting. The bacteria and P. palmivora were originally isolated from tuber rot infected cassava from the field having a disease incidence percentage of above 40%, checked for pathogenicity and were proven for Koch postulate. The detached tubers of disease resistant (cv. Sree Pathmanabha) and susceptible cultivars (cv. M₄) of cassava were used for the study. The effect of bacterial association was studied in Cassava agar plate assay, in detached tubers and in in planta experiments in in vitro glass house conditions. The bacterial isolates were identified as Gram-negative coco bacilli of class Pseudomonas, Erwinia amylovora and Achromobacter denitrificans.     

Poplar stem blister canker and its control strategies by plant extracts

Stem blister canker is a serious stem disease in the Populus genus in China. The pathogen was confirmed as Botryosphaeria dothidea (Moug. ex Fr.) Ces. et de Not. based on its morphological, pathological and molecular features. Seven selected plant species Artemisia annua, Gleditsia sinensis, Lonicera japonica, Macleaya cordata, Phyllodendron chinense, Polygonum cuspidatum and Scutellaria baicalensis, belonging to different families from China, were extracted with the solvent 95% (v/v) ethanol to yield ethanol extracts which were used to evaluate their antifungal activity against poplar stem canker pathogen by a mycelial radial growth inhibition test. Of them, the extracts of M. cordata, P. cuspidatum and S. baicalensis showed stronger antifungal activity, the median inhibitory concentration (IC₅₀) was 0.3125, 1.0219 and 0.9675 mg ml⁻¹ separately. The control effect of extracts of M. cordata, P. cuspidatum and S. baicalensis on poplar stem canker disease was between 62% and 85%. The inhibitory effect of the extracts indicated the importance of some plant species as possible natural source of fungicidal material.     

In vitro standardisation of resistance screening methods in cassava against tuber rot disease

Cassava tuber rot caused by Phytophthora palmivora in growing regions of Tamil Nadu and Kerala, is causing yield loss up to 80%. In the present study, resistance reactions of 10 cassava cultivars were analysed on leaf, stem and tuberous roots by artificial inoculation method in search of a suitable in vitro resistant screening method. Leaf and tuber analysis showed positive correlation (0.883) but the stem-based results showed negative correlation with leaf and tuber analysis. The analysis exhibited the susceptibility of the cassava cultivars against P. palmivora. Leaf analysis was superior in discriminating even small variations in resistance reactions than tuber analysis. The cultivar Sree Padmanabha showed higher resistance than other cultivars and the level of resistance in a cultivar is heritable which could be helpful in breeding programme. Based on the results it can be concluded that leaves of cassava could be used for screening resistance in the host and also in analysing the virulence of the isolate. This is the first report on screening the resistance in cassava cultivars against root rot caused by P. palmivora.     

Heritability of Phenols in the Resistance of Theobroma cacao against Phytophthora megakarya, the Causal Agent of Black Pod Disease

The black pod disease caused by Phytophthora megakarya is responsible for 80% of the cocoa production loss in Cameroon. To assess the resistance of cocoa plants against this pathogen, necrotic lesions, phenolic content and qualitative alteration of phenolics were conducted in ICS84 and ICS95 clones (two Trinitario introduced from Trinidad) and their hybrids (families F30 and F25) derived from reciprocal cross breeding between these two parental clones after inoculation. The existence of strong hybrid vigour has been shown. Ninety percentage of the hybrid's genotypes manifested a positive heterosis effect for the development of lesion size. This suggests the existence of hybrid vigour with a genetic additive effect. F3086, F2509, F2552 and F2586 hybrids were characterized by localized lesions. Those hybrids genotypes can be considered as elite clones. In relation to analysis of total phenolics and lesion size, no maternal effect was detected in the transmission of these characters. A significant and negative correlation (r = −0.683) (P < 0.01) has been observed between necrosis evolution and phenolics accumulation. The values of the heritability of lesion size and the total phenolic content in offsprings did not permit to show the maternal effect. Qualitative analyses of phenolics showed high flavonones content in cocoa leaves. Qualitative analyses of phenolics in ICS84, ICS95 clones and their hybrids showed a modification of the phenolics profiles, notably concerning apigenin and luteolin derivatives due to the inoculation. These compounds, along with others that were not identified, could have a role in the reaction and mechanism of defence of cocoa against P. megakarya.     

Phytophthora Crown and Root Rot of Apricot Caused by Phytophthora palmivora in Turkey

Forty‐nine Phytophthora isolates were obtained from roots and crown of apricot trees with symptoms of decline grown in commercial orchards in Malatya, Elaz?? and Diyarbak?r provinces, Turkey, in 2011 and 2013. All of the recovered isolates were identified as Phytophthora palmivora on the basis of morphological characteristics. Blast analysis of ITS region sequences of rDNA of 5 isolates revealed 100% identity with a reference isolates of P. palmivora from GenBank. Isolates of P. palmivora were pathogenic on 12‐month‐old wild apricot rootstock ‘Zerdali’ plants that were wound inoculated on the roots and on the crown. This study demonstrated that P. palmivora is the cause of the crown and root rot found on apricot in Turkey. To our knowledge, this is the first report of P. palmivora on this host plant.     

Genistein as an endogenous natural substrate of acidic peroxidases in lupin hypocotyls

Crude steam distillate from Ocimum gratissimum sprayed onto infection courts on detached cocoa pods moments after inoculation with Phytophthora palmivora completely inhibited the pathogen and blackpod lesion development on 75% of the infection courts. Disease suppression obtained with the extract was comparable to that obtained with a 2% Kocide 101 suspension. In the field, the O. gratissimum extract also suppressed lesion development although to a significantly lower (P = 0.05) extent in comparison with Kocide 101. Blackpod lesion expansion rates of 3.80, 3.56, 2.71 and 0.78 cm/day, respectively, were associated with pods treated in the field with C. citratus extract, tap water, O. gratissimum extract and 2% Kocide 101. The extract from Cymbopogon citratus was also ineffective on detached pods. Sporangia of P. palmivora from sporu-lating blackpod lesions on both detached and non-detached pods lost their infectivity within 1 h of treatment with the O. gratissimum extract. This effect was superior to that obtained with Kocide 101. Fungitoxicity of the extract on pods, however, was lost within 3 h of application. Thus, despite its in vivo effectiveness as an eradicant, the O. gratissimum extract, in its present form, has limited utility as a protectant fungicide.