VIRUS DISEASES OF CACAO IN WEST AFRICA IX. STRAIN VARIATION AND INTERFERENCE IN VIRUS 1A

Cacao virus iA, the most important and prevalent of the viruses that attack cacao in the Gold Coast, occurs in strains that differ widely in their virulence towards cacao. Outbreaks usually contain trees infected with different strains and individual trees are often infected simultaneously with more than one strain; this can be demonstrated by coppicing the trees, and by inoculating sets of test plants with grafts from different parts of one tree. Neither mild nor virulent strains seemed to be consistently dominant in roots or in other parts of cacao trees.
Cacao plants infected with mild strains were nearly always protected against the effects of infection by virulent strains; however, virulent strains entered hosts already infected with mild strains, but usually without causing any symptoms unless the plants were coppiced. The severe symptoms that developed on new growth from such coppiced plants were seldom repeated in later growth. Mealybugs transmitted the virulent strains from leaves with symptoms characteristic of infection by the latter, but not from leaves free from such symptoms. These results suggest that the multiplication of a virulent strain is impeded in plants infected with a mild strain.
In the field, infection with a mild strain protected mature trees against the effects of virulent strains spread by mealybugs. During 3 years in which 273 out of 387 previously uninfected trees became severely diseased, only 35 out of 416 infected with themildstrain developed symptoms of infectionwiththevirulent strain. Five years after infection with the mild strain, trees were yielding I pod per tree more than in the year they were infected, whereas the decrease on trees infected with the virulent strain was 16 pods per tree. Some limitations in the practical application of protection by mild strains, and objections to its use as a control measure, are discussed.     

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 DISTRIBUTION OF VIRUSES IN DIFFERENT LEAF TISSUES AND ITS INFLUENCE ON VIRUS TRANSMISSION BY APHIDS

Exposing both surfaces of leaves systemically infected with cabbage black ring spot virus (CBRSV) or henbane mosaic virus to ultra-violet radiation decreases the infectivity of expressed sap to about one-fifth. As irradiation probably inactivates virus mainly in the epidermis, which occupies about one-quarter the volume of the leaves, these viruses seem to occur at much higher concentrations in sap from the epidermis than in sap from other cells. By contrast, tobacco mosaic virus seems not to occur predominantly in the epidermis.
CBRSV and henbane mosaic virus are normally transmitted most frequently by previously fasted aphids that feed for only short periods on infected leaves, but aphids treated like this transmit rarely from leaves that have been exposed to ultraviolet radiation. Irradiation has relatively little effect on the proportion of aphids that transmit after long infection feedings. Fasting seems to increase transmission by increasing the probability that aphids will imbibe sap from the epidermis of leaves they newly colonize. With longer periods on infected leaves, the ability of fasted aphids to transmit probably decreases because they then feed from deeper cells and their stylets contain sap with less virus. Only virus contained in the stylets seems to be transmitted, not virus taken into the stomach. About half the transmissions of henbane mosaic virus by aphids that have colonized tobacco leaves for hours may be caused by insects that temporarily cease feeding on the phloem and newly penetrate the epidermis.
Irradiating infected leaves affected the transmission of sugar-beet mosaic virus in the same way as that of henbane mosaic virus, but had little effect on the transmission of beet yellows virus, whose vectors become more likely to transmit the longer they feed on infected plants.     

VIRUS DISEASES OF CACAO IN WEST AFRICA

Of the tested plants which are indigenous to West Africa, three species in the Bombacaceae and four in the Steruliaceae are susceptible to one or more of four viruses from cacao, three occurring in the Gold Coast and one in Nigeria. These species are less affected than cacao by the viruses; some show transient leaf symptoms and others are symptomless carriers. The development of spines on the stems of Ceiba pentandra seedlings is suppressed by infection with virus 1 A.
In general, the indigenous species are more difficult to infect than cacao, and mealybugs do not become infective as readily when feeding on them as when feeding on infected cacao. The availability of the viruses to vectors seems to be correlated with severity of symptoms, and transmission from infected plants to cacao becomes less frequent with increasing duration of infection.
Ceiba pentandra trees were found naturally infected in the Gold Coast and Nigeria. In the Western Province of the Gold Coast, Cola chlamydantha trees in cacao farms and forests were naturally infected with viruses apparently identical with those causing swollen shoot of cacao there.
There is little doubt that C. chlamydantha trees are an important source of virus for cacao trees. Whenever possible these and other alternative hosts growing near to cacao should be destroyed.     

VIRUS DISEASES OF ADANSONIA DIGITATA L. (BOMBACACEAE) AND THEIR RELATION TO CACAO IN GHANA

Virus-infected Adansonia digitata L. were found in the Guinea-savannah woodland forming the Northern Territories and on the Accra plains of Ghana, but those tested in the rain forest were not infected. Three viruses isolated from infected trees resembled the Kpeve cacao virus, but no definite relationship could be established. It is unlikely that the original outbreaks of swollen shoot in cacao came from A. digitata.     

Virus diseases of cacao in West Africa; effect of virus infection on growth and water content of cacao seedlings

Apparently healthy cacao seedlings were compared with those infected before planting with 'swollen shoot' viruses. The leaf area and the fresh and dry weights of each organ were measured. Infected plants were lower in dry weight, leaf area, relative growth rate and net assimilation rate; a smaller proportion of the dry matter was in the leaves and lateral roots, a larger proportion in stems and tap roots. Infection caused extensive necrosis of the lateral roots, and reduced the rate of depletion of reserves in the cotyledons and the water content of the plant. Many of these effects were apparent within a month of infection and planting.     

THE DIFFERENT DISTRIBUTION OF TWO BRASSICA VIRUSES IN THE PLANT AND ITS INFLUENCE ON SPREAD IN THE FIELD

Previous knowledge provided no explanation for the greater prevalence of cauliflower mosaic than of cabbage black ring spot in field crops of cauliflower. Both viruses are spread principally by Myzus persicae and Brevicoryne brassicae , and both are transmitted equally readily from infected seedlings. Cabbage black ring spot virus has a much wider host range, and sap from infected leaves has a higher dilution end-point than sap from leaves infected with cauliflower mosaic virus.
At least part of the difference between the rate at which the two viruses spread in the field may be accounted for by the different manner in which they are distributed in old infected plants, and the effect this has on transmission by aphids. Cauliflower mosaic virus occurs in high concentration in all the new leaves produced by infected plants. Cabbage black ring spot virus, on the other hand, occurs mainly in the older leaves, and even there is localized in parts that show symptoms. Only in recently infected plants does cabbage black ring spot virus occur in young leaves.
After flying, most aphids alight on the upper parts of plants; they are therefore less likely to acquire cabbage black ring spot virus than cauliflower mosaic virus. It may be significant that cabbage, a host in which old leaves are in a more favourable position for alighting aphids than are those of cauliflower, is also often extensively infected with cabbage black ring spot virus.     

STUDIES ON THE HOST RANGES OF VIRUSES FROM THEOBROMA CACAO L.

Only plants in the natural order Tiliales were susceptible to experimental infection by viruses found infecting cacao. Of sixty-three species in the Tiliales colonized with infective mealybugs, twenty-eight became infected with the New Juaben virus. The ease with which different species contracted infection and the severity of symptoms varied considerably. The host ranges of seven other viruses were all contained in that of the New Juaben virus. The viruses could be divided into three groups according to their host ranges, and it is suggested that host ranges may prove helpful in attempts to classify cacao viruses. Four species of Corchorus were all rapidly killed by any of the eight viruses.     

SOME EFFECTS OF VIRUS INFECTION ON LEAF WATER CONTENTS OF NICOTIANA SPECIES

Infection with tobacco mosaic virus decreases the water content which detached tobacco leaves attain when kept for 20 hr. in conditions of minimum water stress, and does so more when the plants are kept in light before inoculation than when they are kept in darkness. No such effects of infection during the first day after inoculation were obtained with tobacco leaves infected with either tobacco etch virus or potato virus X , or with Nicotiana glutinosa leaves infected with tobacco mosaic virus. These results, like those showing early effects of TMV on respiration and photosynthesis of tobacco leaves, suggest that inoculation with TMV affects deeper leaf tissues than the epidermis earlier in tobacco leaves than in other leaves, and earlier than other viruses in tobacco leaves.     

TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4

The interaction amongst papain-like cysteine-proteases (PLCP) and their substrates and inhibitors, such as cystatins, can be perceived as part of the molecular battlefield in plant-pathogen interaction. In cacao, four cystatins were identified and characterized by our group. We identified 448 proteases in cacao genome, whereof 134 were cysteine-proteases. We expressed in Escherichia coli a PLCP from cacao, named TcCYSPR04. Immunoblottings with anti-TcCYSPR04 exhibited protein increases during leaf development. Additional isoforms of TcCYSPR04 appeared in senescent leaves and cacao tissues infected by Moniliophthora perniciosa during the transition from the biotrophic to the saprophytic phase. TcCYSPR04 was induced in the apoplastic fluid of Catongo and TSH1188 cacao genotypes, susceptible and resistant to M. perniciosa, respectively, but greater intensity and additional isoforms were observed in TSH1188. The fungal protein MpNEP induced PLCP isoform expression in tobacco leaves, according to the cross reaction with anti-TcCYSPR04. Several protein isoforms were detected at 72 hours after treatment with MpNEP. We captured an active PLCP from cacao tissues, using a recombinant cacao cystatin immobilized in CNBr-Sepharose. Mass spectrometry showed that this protein corresponds to TcCYSPR04. A homology modeling was obtained for both proteins. In order to become active, TcCYSPR04 needs to lose its inhibitory domain. Molecular docking showed the physical-chemical complementarities of the interaction between the cacao enzyme and its inhibitor. We propose that TcCYSPR04 and its interactions with cacao cystatins are involved in the senescence and necrosis events related to witches’ broom symptoms. This molecular interaction may be the target for future interventions to control witches'' broom disease.     

NOTES ON A VIRUS DISEASE OF CACAO

A virus disease of cacao, discovered in Trinidad towards the end of 1943, is apparently confined to the north-western corner of the is land and does not occur in the main cacaogrowing areas of eastern, central and southern Trinidad; nor has it been found in Venezuela, Tobago or the Windward Islands of Grenada, St Vincent, St Lucia and Dominica. The symptoms resemble those of West African swollen shoot disease, consisting of vein-clearing or mosaic of the leaves and red-mottle of the leaves and pods. No swellings are formed, but recent observations indicate that the disease may cause both dieback and reduction in yield. There are two strains closely resembling each other. The spread of the disease under field conditions has been demonstrated, and although the rate of spread has varied somewhat, a mean increase of 41 % over the original number of infected trees has been found over a 10-month period. As with swollen shoot disease, existing diseased areas have increased in size and new outbreaks at short distances have occurred. Both strains were readily transmitted by budding, the incubation period varying from 34 to 136 days, with a mean around 90 days, but no insect vectors have been found. There is at present little reason to believe that any varieties of cacao are immune to the disease or are even symptomless carriers. The question of eradicating this virus, while its distribution remains limited, should be seriously considered.     

Microbiology of the root region of coconut and cacao under mixed cropping

Summary Mixed cropping of cacao in coconut plantations improved the microbial activity in the rhizosphere of coconut which may be attributed to an increase in organic matter content of soil due to periodic shedding of cacao leaves. When compared to coconut cultivation without cacao, mixed cropping of coconut with cacao appeared to stimulate the population of bacteria and fungi in the rhizosphere of coconut, including the nitrogen fixing and phosphate solubilising bacteria. The occurrence of indole acetic acid producing Escherichia sp. on the root surface of coconut and the appearance of gibberellin(like-substance) producing Aspergillus flavus and A. fumigatus in the rhizosphere were other salient observations made in a pioneering study on the microbiology of the root region of plantation crops under mixed cropping. re]19750702     

Peroxidase Activity and Isoperoxidase Pattern in Tobacco Leaves Infected with Tobacco Necrosis Virus and Other Viruses Inducing Necrotic and Non-Necrotic Alterations

The level of peroxidase activity was greatly enhanced in tobacco leaves infected by tobacco necrosis virus (TNV) and other viruses which induce necrotic symptoms (TMV, ToMV and PVY^N). The intensity was related to the age of the leaves infected: absent or neglible in mature leaves and very pronounced in young growing infected leaves. On the contrary, changes in peroxidase activity were negligible when the infection was provoked by viruses which do not produce necrotic reactions (TMV and PVY^O). Analysis of the peroxidase isoenzymes, pattern in tobacco leaves infected by TNV and other necrosis-inducing viruses revealed in all cases, a slight increase in anionic (pl 3.5–3.7) and a considerable increase in moderately anionic isoenzymes particularly the pl 4.6 isoenzyme which in TNV and PVY^N-infected leaves reached levels up to 21 and 72 times the healthy control values. A considerable increase in the cationic (pl9.3–8.8) isoenzymes and the appearance of one moderately cationic isoenzyme (pl 8.2) was also detected. In leaf extracts from-virus-infected tobacco leaves with nonnecrotic response, no, or negligible alterations on the isoenzyme pattern were detected. However, infection by a fungal parasite (Erisyphe cichoracearum), which established a fully compatible, non-necrotic, interaction with tobacco leaves, like the necrosis-inducing viruses, changed the isoperoxidase pattern. The data suggest the necrotic alterations and associated changes in the peroxidase activity and isoperoxidase pattern in virus-infected leaves are not clearly related.     

THE INFECTION OF PLANTS BY VIRUSES THROUGH ROOTS

Roots of young tomato plants became infected when inoculated with tomato bushy stunt, tobacco mosaic, and potato X viruses. Root infections also occurred when these viruses were added to soil or culture solutions in which plants were growing.
The viruses were sometimes localized around their initial entry points in roots; sometimes they invaded the root system but not the shoots, and sometimes they produced full systemic infection of roots and shoots. In some experiments, but not all, systemic infections were more frequent when the upper tap root or superficial roots were inoculated than when fibrous roots were inoculated.
In both tomato and potato, virus X spread from diseased to healthy plants sharing the same culture solution, if their roots were in contact, but not otherwise. Infection of the roots of potato plants by inoculation, produced only one plant with virus-infected haulms, although several had infected tubers.     

PRIMULA OBCONICA, A CARRIER OF TOBACCO NECROSIS VIRUSES

A tobacco necrosis virus has been isolated from the leaves and flowers of naturally infected Primula obconica plants. Although the virus produces no necrotic symptoms, it is not distributed uniformly through primulas, but occurs only in isolated regions, most of the tissues being apparently virus-free.
When inoculated to healthy primulas, three tobacco necrosis viruses were found to behave similarly. They all enter and multiply locally, but produce no symptoms; movement from the inoculated areas occurs only rarely and then does not cause a full systemic infection but only further localized infections. Multiplication of the viruses in primula is slower than in tobacco or French bean, which react necrotically.
The uncertainties in interpreting results of tests for tobacco necrosis viruses are described and possible explanations of natural infections are discussed.     

THE MANNER OF TRANSMISSION OF SOME BARLEY YELLOW-DWARF VIRUSES BY DIFFERENT APHID SPECIES

Some barley yellow-dwarf (BYD) viruses isolated from cereal crops in Great Britain were transmitted by Rhopalosiphum padi , L. and others were not. Sitobion fragariae (Walker), S. avenae (Fabricius), and Metopolophium dirhodum (Walker) all transmitted viruses of both types, but they usually transmitted those of which Rhopalosiphum was a vector less readily than did R. padi. The transmissibility of a virus by a given aphid species was not affected by transmission with another, less efficient, vector species. Neomyzus circumflexus (Buckt.) and Rhopalosiphum maidis (Fitch) transmitted the few viruses with which they were tested.
A few R. padi acquired virus from infected leaves during 30 min. feeding and inoculated healthy seedlings during 15 min. feeding, but the minimum total time taken to acquire and transmit was 10 hr. and 32 hr. were needed for about half the aphids that were able to acquire and transmit virus to do so. This may indicate the existence of a short latent period of the virus in the vector, although the evidence is not conclusive. The times spent on infected plants influenced the results more than those spent on healthy ones; many transmissions occurred with short feeding times on healthy plants so long as the time spent on infected leaves was long, but the reverse was not true. Nymphs of R. padi that moulted after they left infected plants on which they fed long enough to become infective, infected slightly fewer plants than adults fed for the same times.     

Biochemical characterization of enoyl reductase involved in Type II fatty acid synthesis in the intestinal coccidium Eimeria tenella (Phylum Apicomplexa)

Moniliophthora perniciosa is the causative agent of witches' broom disease in Theobroma cacao. Exogenously provided abscisic acid (ABA), indole-3-acetic acid (IAA), jasmonic acid (JA), and salicylic acid (SA) promoted mycelial growth, suggesting the ability of the pathogen to metabolize plant hormones. ABA, IAA, JA, and SA were found endogenously in the mycelium and in the fruiting body of the pathogen. The pathogen contained high amounts of SA in the mycelium (0.5+/-0.04 microg g(-1) DW) and IAA (2+/-0.6 microg g(-1) DW) in the basidiocarps. Growth of the mycelium in the presence of host leaves for 10 days did not affect ABA or JA content of the leaves but IAA and SA increased 2.5- and 11-fold, respectively. The amounts of IAA and SA in infected leaves increased beyond the levels of the uninfected leaves and suggest a synergistic response to host-pathogen interaction. The ability of M. perniciosa to produce and sustain growth in the presence of elevated endogenous IAA and SA levels during colonization indicates that these phytohormones contribute to its pathogenicity.     

The Influence of Plant Water Deficit on Distribution of 14C-labelled Assimilates in Cacao Seedlings

The relationship between plant water status and distributionof ¹⁴C-labelled assimilates in cacao (Theobroma cacao L.) wasevaluated after ¹⁴CO₂ pulse labelling leaves of seedlings subjectedto varying levels of water deficiency. The proportion of ¹⁴Cexported by source leaves was strongly affected by seedlingwater status. An increasing proportion of labelled assimilatesremained in source leaves at both 24-h and 72-h harvests aswater stress intensity increased. Water stress reduced the distributionof exported label to leaves and to the expanding flush in particularbut increased the proportion of label in stems and roots. Theresults suggest that current photoassimilates may be temporarilystored in source leaves and stems of cacao seedlings duringperiods of plant water deficit. The stress-induced changes inpartitioning of labelled carbon were in concordance with changesin shoot to root biomass ratios, which was likely due to greaterreduction in growth of above-ground organs to that of roots. Theobroma cacao L, assimilate partitioning, cacao, ¹⁴C-photoassimilate, water stress, water potential     

Colletotrichum gloeosporioides s.l. associated with Theobroma cacao and other plants in Panama: multilocus phylogenies distinguish host-associated pathogens from asymptomatic endophytes

Colletotrichum interacts with numerous plant species overtly as symptomatic pathogens and cryptically as asymptomatic endophytes. It is not known whether these contrasting ecological modes are optional strategies expressed by individual Colletotrichum species or whether a species' ecology is explicitly pathogenic or endophytic. We explored this question by inferring relationships among 77 C. gloeosporioides s.l. strains isolated from asymptomatic leaves and from anthracnose lesions on leaves and fruits of Theobroma cacao (cacao) and other plants from Panamá. ITS and 5'-tef1 were used to assess diversity and to delineate operational taxonomic units for multilocus phylogenetic analysis. The ITS and 5'-tef1 screens concordantly resolved four strongly supported lineages, clades A-D: Clade A includes the ex type of C. gloeosporioides, clade B includes the ex type ITS sequence of C. boninense, and clades C and D are unidentified. The ITS yielded limited resolution and support within all clades, in particular the C. gloeosporioides clade (A), the focal lineage dealt with in this study. In contrast the 5'-tef1 screen differentiated nine distinctive haplotype subgroups within the C. gloeosporioides clade that were concordant with phylogenetic terminals resolved in a five-locus nuclear phylogeny. Among these were two phylogenetic species associated with symptomatic infections specific to either cacao or mango and five phylogenetic species isolated principally as asymptomatic infections from cacao and other plant hosts. We formally describe two new species, C. tropicale and C. ignotum, that are frequent asymptomatic associates of cacao and other Neotropical plant species, and epitypify C. theobromicola, which is associated with foliar and fruit anthracnose lesions of cacao. Asymptomatic Colletotrichum strains isolated from cacao plants grown in China included six distinct C. gloeosporioides clade taxa, only one of which is known to occur in the Neotropics.     

Comparative expression profiling of Nicotiana benthamiana leaves systemically infected with three fruit tree viruses

Plant viruses cause a wide array of disease symptoms and cytopathic effects. Although some of these changes are virus specific, many appear to be common even among diverse viruses. Currently, little is known about the underlying molecular determinants. To identify gene expression changes that are concomitant with virus symptoms, we performed comparative expression profiling experiments on Nicotiana benthamiana leaves infected with one of three different fruit tree viruses that produce distinct symptoms: Plum pox potyvirus (PPV; leaf distortion and mosaic), Tomato ringspot nepovirus (ToRSV; tissue necrosis and general chlorosis), and Prunus necrotic ringspot ilarvirus (PNRSV; subtle chlorotic mottling). The numbers of statistically significant genes identified were consistent with the severity of the observed symptoms: 1,082 (ToRSV), 744 (PPV), and 89 (PNRSV). In all, 56% of the gene expression changes found in PPV-infected leaves also were altered by ToRSV, 87% of which changed in the same direction. Both PPV- and ToRSV-infected leaves showed widespread repression of genes associated with plastid functions. PPV uniquely induced the expression of large numbers of cytosolic ribosomal genes whereas ToRSV repressed the expression of plastidic ribosomal genes. How these and other observed expression changes might be associated with symptom development are discussed.