The limited host range of an Agrobacterium tumefaciens strain extended by a cytokinin gene from a wide host range T-region

The host range of Agrobacterium tumefaciens strain LBA649 (pTiAg57) is limited to grapevine and a few other plant species. Its host range was extended through the introduction of the T-region from the wide host range octopine plasmid pTiAch5. In contrast, R prime plasmids harboring the entire wide host range virulence region were unable to achieve this effect. Via site-directed mutagenesis a search was performed to identify the T-DNA genes which were responsible for the observed host range extension. Inactivation of one of the onc-genes (the cyt gene) was found to abolish the capacity of the T-region to extend the host range of LBA649. Therefore, we cloned the cyt gene into a disarmed T-region plant vector and used it in complementation studies with pTiAg57 via the binary vector strategy. We show that the mere presence of the cyt gene from a wide host range Ti plasmid is sufficient to extend the host range of LBA649 to certain plants. We conclude that the limited host range of LBA649 is not caused by a lack of recognition of plants but is mainly due to the absence or inactivity of a cyt gene in the T-region of pTiAg57.     

Amphotropic retrovirus vector system for human cell gene transfer.

Retroviral vectors have been constructed for gene transfer in mammalian and avian cells, however most retroviral vector systems are complicated by the spread of a replication-competent helper virus. This problem has been circumvented by segregating the viral genome into cis- and trans-acting components. By establishing helper cell lines that produce the trans-acting viral gene products, one can propagate the cis-acting component in them and harvest defective viral particles that contain only the cis-acting component. The cis-acting component can provide a useful vehicle for the highly efficient transfer of genes into target cells. The defective vector systems described to date, however, are restricted in host range to murine, avian, rat, and dog cells. We describe a helper-free vector system based entirely on an amphotropic murine virus with a wide mammalian host range, including the ability to carry out efficient gene transfer into human cells. We also describe a double mutation constructed in the trans-acting genome which reduces the frequency of replication-competent recombinant viruses to undetectable levels.     

FURTHER OBSERVATIONS ON THE PATHOGENICITY OF CALONECTRIA RIGIDIUSCULA (BERK. & BR.) SACC. TO THEOBROMA CACAO L

Quantitative data are provided for the occurrence of Calonectria rigidiuscula and other fungi in occluded Mirid lesions and other sites in shoots of cacao plants. Mirid lesions appear to be particularly favourable for the development of C. rigidiuscula. The fungus also occurs as a saprophyte on cacao pods, and as a wound parasite in woody plants other than cacao; it was successfully introduced into plants of varied affinities by inoculation. C. rigidiuscula spreads from inoculations in cacao stems much more rapidly than other fungi. The results confirm that it is the most important fungus infecting Mirid lesions and causing dieback of cacao in West Africa.
Inoculation tests with a range of Amelonado and Trinitario clones suggested that the clones vary in their susceptibility to C. rigidiuscula , but the plant-to-plant variation was too great to conclude that any one is highly resistant. Various types of introduced cacao were also tested; preliminary experiments indicate that certain types of Upper Amazon cacao may be resistant, but they need further investigation.     

Use of an exotic host plant shifts immunity,chemical defense,and viral burden in wild populations of a specialist insect herbivore

Defense against natural enemies constitutes an important driver of herbivore host range evolution in the wild. Populations of the Baltimore checkerspot butterfly, Euphydryas phaeton (Nymphalidae), have recently incorporated an exotic plant, Plantago lanceolata (Plantaginaceae), into their dietary range. To understand the tritrophic consequences of utilizing this exotic host plant, we examined immune performance, chemical defense, and interactions with a natural entomopathogen (Junonia coenia densovirus, Parvoviridae) across wild populations of this specialist herbivore. We measured three immune parameters, sequestration of defensive iridoid glycosides (IGs), and viral infection load in field‐collected caterpillars using either P. lanceolata or a native plant, Chelone glabra (Plantaginaceae). We found that larvae using the exotic plant exhibited reduced immunocompetence, compositional differences in IG sequestration, and higher in situ viral burdens compared to those using the native plant. On both host plants, high IG sequestration was associated with reduced hemocyte concentration in the larval hemolymph, providing the first evidence of incompatibility between sequestered chemical defenses and the immune response (i.e., the “vulnerable host” hypothesis) from a field‐based study. However, despite this negative relationship between IG sequestration and cellular immunity, caterpillars with greater sequestration harbored lower viral loads. While survival of virus‐infected individuals decreased with increasing viral burden, it ultimately did not differ between the exotic and native plants. These results provide evidence that: (1) phytochemical sequestration may contribute to defense against pathogens even when immunity is compromised and (2) herbivore persistence on exotic plant species may be facilitated by sequestration and its role in defense against natural enemies.     

Molecular Characterization of Cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis>) Germplasm from Jamaica Using Single Nucleotide Polymorphism (SNP) Markers

Cacao is an economically important commodity in Jamaica. Knowledge of the genetic diversity of Jamaican cacao germplasm is essential for their conservation and management. In spite of cacao’s economic importance in Jamaica, the crop is under studied, therefore limiting sound decisions toward improving productivity. Assessment of germplasm and on-farm genetic diversity is required to assist selecting superior genotypes to propagate and distribute across the island, as well as to use them as parental clones in breeding programs. Using 94 single nucleotide polymorphism (SNP) markers, 140 Jamaican cacao samples from two germplasm collections and a farmer’s estate along with 150 reference samples were analyzed. The principal coordinate analysis demonstrated that the majority of the Jamaican cacao selections were hybrids derived from five original germplasm groups, including Criollo, Amelonado and three Upper Amazon Forastero groups. Among the Upper Amazon groups, the Bayesian clustering analysis revealed that the Parinari (PA) ancestral lineage contributed the most (29.9%) to the Jamaican cacao germplasm. The germplasm collections showed greater diversity in terms of ancestral contributions compared to the farmer’s estate. However, the genetic differentiation between the three collecting sites was small (Fst?=?0.036), indicating that samples collected from the three sites were derived from a common pool of germplasm. The current study supports the historical records and clarified the ancestry of Jamaican cacao. Although the majority of the cacao genetic groups were observed in the Jamaican cacao collections, several diversity gaps were found in both germplasm collections and in the farmer’s estate, especially germplasm with disease resistance to cacao frosty pod rot that was recently found in Jamaica.     

Decreased symbiotic effectiveness of Rhizobium leguminosarum strains carrying plasmid RP4

The presence of derivatives of the broad host range plasmid RP4 in strains of Rhizobium leguminosarum biovar viciae severely inhibited nitrogen fixation by these strains in nodules on cultivars of pea (Pisum sativum). The strains formed small white nodules. Yield and total nitrogen values were comparable with those obtained for plants inoculated with a non-nodulating mutant. Strains carrying the same derivatives gave rise to nitrogen fixing nodules when inoculated on cultivars of lentils (Lens culinaris). Similar results were observed with plasmid R702 but not with R751, suggesting that the effect is limited to plasmids of the IncPα classification. Histological examination of nodules induced by strains carrying RP4 indicated that there are fewer infected cells and starch granules are organised unusually in the infected cells. Tn5 mutagenesis of plasmid RP4-4 was undertaken and Tn5 inserts were screened for abolition of the effect on nitrogen fixation. Eight mutants, having no effect on nitrogen fixation, were isolated. Seven of these had lost the ability to transfer by conjugation and the eighth was greatly reduced in conjugation frequency. Physical analysis of the transposon inserts revealed that they were located in the Tra regions of RP4.     

Acquisition of Host Cell DNA Sequences by Baculoviruses: Relationship Between Host DNA Insertions and FP Mutants of Autographa californica and Galleria mellonella Nuclear Polyhedrosis Viruses

Mutants of Autographa californica and Galleria mellonella nuclear polyhedrosis viruses, which produce an altered plaque phenotype as a result of reduced numbers of viral occlusions in infected cells, were isolated after passage in Trichoplusia ni (TN-368) cells. These mutants, termed FP (few-polyhedra) mutants, had acquired cell DNA sequences ranging from 0.8 to 2.8 kilobase pairs in size. The insertions of cell DNA occurred in a specific region between 35.0 and 37.7 map units of the A. californica viral genome. A cloned viral fragment containing one of the host DNA inserts was homologous to host DNA inserts in two other mutant viruses and to dispersed, repetitious sequences in T. ni cell DNA. Most of the homology between the cloned insert and cell DNA was contained within a 1,280-base-pair AluI fragment. Marker rescue studies and analysis of infected-cell-specific proteins suggested that the insertion of cell DNA into the viral genomes resulted in the FP plaque phenotype, possibly through the inactivation of a 25,000-molecular-weight protein.     

A Dual Role for Microbial Pathogen-Derived Effector Proteins in Plant Disease and Resistance

Many proteins from plant pathogens affecting the interaction with the host plant have dual functions: they promote virulence on the host species and they function as avirulence determinants by eliciting defense reactions in host cultivars expressing the appropriate resistance genes. In viruses all proteins encoded by the small genomes can be expected to be essential for viral development in the host. However, in different plants surveillance systems have evolved that are able to recognize most of these proteins. Bacteria and fungi have specialized pathogenicity and virulence genes. Many of the latter were originally identified through the resistance gene-dependent elicitor activity of their products. Their role in virulence only became apparent when they were inactivated or transferred to different microbes or after their ectopic expression in host plants. Many microbes appear to maintain these genes despite their disadvantageous effect, introducing only few mutations to abolish the interaction of their products with the plant recognition system. This has been interpreted as been indicative of a virulence function of the gene products that is not impaired by the mutations. Alternatively, in particular in bacteria there is now evidence that pathogenicity was acquired through horizontal gene transfer. Genes supporting virulence in the donor organism's original host appear to have traveled along. Being gratuitous in the new situation, they may have been inactivated without loss of any beneficial function for the pathogen.     

Herbivory by Strongylocoris leucocephalus (Hemiptera: Miridae) on a novel host plant Adenophora triphylla var. japonica in Japan

When monophagous and oligophagous insect herbivores colonize new areas, they sometimes use novel hosts. The availability of the new hosts can facilitate further expansion into regions beyond the geographic range of the original hosts, resulting in specialization on different host plant taxa in different parts of the herbivore's geographic range. Strongylocoris leucocephalus Linnaeus (Hemiptera: Miridae), which is a plant bug attacking Campanula spp., is widely distributed in the Eurasian continent, North Africa, and northern Japan. In the process of geographic range expansion, S. leucocephalus may incorporate novel host plants into their host range. I investigated the host plant species of S. leucocephalus in central Japan. Field observations revealed that the plant bug fed on the novel host plant Adenophora triphylla (Thunb.) A. DC. var. japonica (Regel) H. Hara (Campanulaceae), although feeding by S. leucocephalus was not associated with plant mortality. However, S. leucocephalus never fed on Campanula punctata Lam. var. hondoensis (Kitam.) Ohwi, the only Campanula species at the study site. The plant bug S. leucocephalus and the novel host plant A. triphylla var. japonica are not entirely distributed sympatrically, suggesting that the plant bug has expanded its geographic range by incorporating new hosts.     

Detection of T-DNA transfer to plant cells by A. tumefaciens virulence mutants using agroinfection

Summary To test whether virulence mutants of Agrobacterium tumefaciens are capable of promoting T-DNA transfer into plant cells, a tandem array of Cauliflower Mosaic Virus (CaMV) DNA was cloned between T-region border sequences on a wide host range plasmid and introduced into various virulence mutants. The resulting strains were used to infect Brassica rapa cv. Just Right. This assay, recently referred to as agroinfection, is based on the appearance of viral symptoms following transfer of T-DNA to plant cells, and is shown to be at least 100 times more sensitive in detecting T-DNA transfer than tumour formation. Mutants in the loci vir A, B and G, which were avirulent on turnip, failed to induce virus symptoms. Of the two vir D mutants tested, neither induced tumours, but one was capable of inducing virus symptoms. Mutants in vir E, C and F, which induced respectively no, small and normal tumours on turnip, all induced virus symptoms.     

Host-Specific Parvovirus Evolution in Nature Is Recapitulated by In Vitro Adaptation to Different Carnivore Species

Canine parvovirus (CPV) emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV), a parvovirus of cats and related carnivores. Although both viruses have wide host ranges, analysis of viral sequences recovered from different wild carnivore species, as shown here, demonstrated that >95% were derived from CPV-like viruses, suggesting that CPV is dominant in sylvatic cycles. Many viral sequences showed host-specific mutations in their capsid proteins, which were often close to sites known to control binding to the transferrin receptor (TfR), the host receptor for these carnivore parvoviruses, and which exhibited frequent parallel evolution. To further examine the process of host adaptation, we passaged parvoviruses with alternative backgrounds in cells from different carnivore hosts. Specific mutations were selected in several viruses and these differed depending on both the background of the virus and the host cells in which they were passaged. Strikingly, these in vitro mutations recapitulated many specific changes seen in viruses from natural populations, strongly suggesting they are host adaptive, and which were shown to result in fitness advantages over their parental virus. Comparison of the sequences of the transferrin receptors of the different carnivore species demonstrated that many mutations occurred in and around the apical domain where the virus binds, indicating that viral variants were likely selected through their fit to receptor structures. Some of the viruses accumulated high levels of variation upon passage in alternative hosts, while others could infect multiple different hosts with no or only a few additional mutations. Overall, these studies demonstrate that the evolutionary history of a virus, including how long it has been circulating and in which hosts, as well as its phylogenetic background, has a profound effect on determining viral host range.     

Dispersal of a Human-Cultivated Crop by Wild Chimpanzees (<Emphasis Type="Italic">Pan troglodytes verus</Emphasis>) in a Forest–Farm Matrix

With the conversion of natural habitats to farmland, nonhuman primates (hereafter primates) are increasingly exposed to agricultural crops. Although frugivorous primates are important seed dispersers that sometimes feed on agricultural fruits, evidence for dispersal of crops by primates is lacking. Here, we examine flexible feeding on cacao (Theobroma cacao) fruit and seed dispersal patterns by chimpanzees (Pan troglodytes verus) at Bossou in Guinea, and consequent cacao germination and survival. From direct observations, we confirm that cacao fruit is not an important food to chimpanzees, representing 0.23 % of focal animal feeding time. Chimpanzees ingest cacao pulp and either spit out the large seeds intact from unripe cacao fruit or swallow the seeds from ripe cacao fruits, which are consequently deposited in feces. From ecological surveys we show that chimpanzees distributed cacao extensively throughout their home range, at a mean distance of 407 m?±?SE 0.6 (N?=?90 clusters, range: 4–1130 m) from cacao plantations. As distance from the cacao plantation increased, cacao plants were more likely to survive. Other factors, including number of cacao plants in a cluster, plant height, and openness of the understory did not predict short-term cacao survival. Cacao plants within the forest did not produce fruit. By contrast, when chimpanzees deposited seeds in a plantation, cacao plants produced fruits as a result of farmers’ maintenance of the area. Our local-scale findings emphasize the complex behavioral and ecological interconnections between coexisting humans and primates in agricultural landscapes and generate interesting questions regarding primate niche construction and crop “ownership” related to who “plants” the crop.     

Retroviral DNA Integration

DNA integration is a unique enzymatic process shared by all retroviruses and retrotransposons. During integration, double-stranded linear viral DNA is inserted into the host genome in a process catalyzed by the virus-encoded integrase (IN). The mechanism involves a series of nucleophillic attacks, the first of which removes the terminal 2 bases from the 3′ ends of the long terminal repeats and of the second which inserts the viral DNA into the host genome. IN specifically recognizes the DNA sequences at the termini of the viral DNA, juxtaposing both ends in an enzyme complex that inserts the viral DNA into a single site in a concerted manner. Small duplications of the host DNA, characteristic of the viral IN, are found at the sites of insertion. At least two host proteins, HMG-I(Y) and BAF, have been shown to increase the efficiency of the integration reaction.     

Endemic predators, invasive prey and native diversity

Interactions between native diversity and invasive species can be more complex than is currently understood. Invasive ant species often substantially reduce diversity in the native ants diversity that act as natural control agents for pest insects. In Indonesia (on the island of Sulawesi), the third largest cacao producer worldwide, we show that a predatory endemic toad (Ingerophrynus celebensis) controls invasive ant (Anoplolepis gracilipes) abundance, and positively affects native ant diversity. We call this the invasive-naivety effect (an opposite of enemy release), whereby alien species may not harbour anti-predatory defences against a novel native predator. A positive effect of the toads on native ants may facilitate their predation on insect vectors of cacao diseases. Hence, toads may increase crop yield, but further research is needed on this aspect. Ironically, amphibians are globally the most threatened vertebrate class and are strongly impacted by the conversion of rainforest to cacao plantations in Sulawesi. It is, therefore, crucial to manage cacao plantations to maintain these endemic toads, as they may provide critical ecosystem services, such as invasion resistance and preservation of native insect diversity.     

Molecular characterization of an earliest cacao (Theobroma cacao L.) collection from Upper Amazon using microsatellite DNA markers

Cacao (Theobroma cacao L.) is indigenous to the Amazon region of South America. The river basins in the Upper Amazon harbor a large number of diverse cacao populations. Since the 1930s, several numbers of populations have been collected from the present-day Peruvian Amazon and maintained as ex situ germplasm repositories in various countries, with the largest held in the International Cacao Genebank in Trinidad. The lack of information on population structure and pedigree relationship and the incorrect labeling of accessions are major concerns for efficient conservation and use of cacao germplasm. In the present study, we assessed the individual identity, sibship, and population structure in cacao populations collected from the present-day Loreto Region, Peru in the 1930–1940s. Using a capillary electrophoresis genotyping system, we analyzed the simple sequence repeat variation of 612 cacao accessions collected from the Marañon, Nanay, and Ucayali river systems. A total of 180 cases of mislabeling were identified using a Bayesian clustering method for admixture detection. Using maximum likelihood-based methods, we reconstructed 78 full-sib families nested in 48 half-sib families, indicating that the pods collected in the 1930s were from 48 mother trees, maximum. Likelihood simulation also identified eight probable parents that are responsible for 117 pairs of mother–offspring relationships in this collection. Principal coordinate analysis (PCoA) and the Bayesian clustering method cohesively demonstrated a pronounced structure of genetic diversity, stratified by the river systems of the Peruvian Amazon. Our results also show that, in spite of the high level of allelic diversity in this collection, it was composed of a large number of related family members collected from a relatively small area, including a couple of sites in the Ucayali and Nanay rivers, as well as the lower Marañon river near Iquitos. The vast majority of the Peruvian Amazon, especially the upper Marañon River and its tributaries, have not been sampled by collecting expeditions. The improved understanding of the individual identities, genealogical relationships, and geographical origin of cacao germplasm in this collection will contribute to more efficient conservation and utilization of these germplasm. Additionally, this study also provides more baseline information to help guide future collecting expeditions in the Peruvian Amazon.     

Genetic Structure and Molecular Diversity of Cacao Plants Established as Local Varieties for More than Two Centuries: The Genetic History of Cacao Plantations in Bahia,Brazil

Bahia is the most important cacao-producing state in Brazil, which is currently the sixth-largest country worldwide to produce cacao seeds. In the eighteenth century, the Comum, Pará and Maranhão varieties of cacao were introduced into southern Bahia, and their descendants, which are called ‘Bahian cacao’ or local Bahian varieties, have been cultivated for over 200 years. Comum plants have been used to start plantations in African countries and extended as far as countries in South Asia and Oceania. In Brazil, two sets of clones selected from Bahian varieties and their mutants, the Agronomic Institute of East (SIAL) and Bahian Cacao Institute (SIC) series, represent the diversity of Bahian cacao in germplasm banks. Because the genetic diversity of Bahian varieties, which is essential for breeding programs, remains unknown, the objective of this work was to assess the genetic structure and diversity of local Bahian varieties collected from farms and germplasm banks. To this end, 30 simple sequence repeat (SSR) markers were used to genotype 279 cacao plants from germplasm and local farms. The results facilitated the identification of 219 cacao plants of Bahian origin, and 51 of these were SIAL or SIC clones. Bahian cacao showed low genetic diversity. It could be verified that SIC and SIAL clones do not represent the true diversity of Bahian cacao, with the greatest amount of diversity found in cacao trees on the farms. Thus, a core collection to aid in prioritizing the plants to be sampled for Bahian cacao diversity is suggested. These results provide information that can be used to conserve Bahian cacao plants and applied in breeding programs to obtain more productive Bahian cacao with superior quality and tolerance to major diseases in tropical cacao plantations worldwide.     

Genetic identity and diversity of Nigerian cacao genebank collections verified by single nucleotide polymorphisms (SNPs): a guide to field genebank management and utilization

Nigeria is the sixth largest cacao producer in the world. Field performance and quality of cacao hybrid families is largely dependent on the genetic integrity of parental clones obtained in field genebank collections. However, information on the impact of mislabeling on seed garden output in Nigeria is lacking. Using 63 single nucleotide polymorphism (SNP) markers, we analyzed 1457 cacao trees sampled from seven major field genebank plots in Nigeria to assess the genetic integrity in Nigerian cacao germplasm. The procedure of multilocus matching with known reference clones revealed up to 78% mislabeling in recently introduced international germplasm. A high rate of mislabeling was also revealed in the West African local selections and breeding lines, using Bayesian assignment test. The problem of mislabeling has been attributed to errors from the sources of introduction, pre-planting labeling errors, and rootstocks overtaking budded scions due to poor field management. The analysis of genetic diversity revealed a good representation of the available cacao germplasm groups in Nigerian field genebanks, indicating that the genetic base of Nigeria cacao germplasm has been significantly widened through germplasm introductions. However, only a small proportion of the available germplasm in the genebank have been utilized for variety development. This study proved the utility of SNP markers for cleaning up the genebanks and reducing offtypes; thereby providing a strong basis for improving the accuracy and efficiency in cacao genebank management and breeding, as well as for mobilizing improved varieties to cacao farmers in Nigeria.     

The genome of ‘Candidatus Phytoplasma solani’ strain SA-1 is highly dynamic and prone to adopting foreign sequences

Bacteria of the genus ‘Candidatus Phytoplasma’ are uncultivated intracellular plant pathogens transmitted by phloem-feeding insects. They have small genomes lacking genes for essential metabolites, which they acquire from either plant or insect hosts. Nonetheless, some phytoplasmas, such as ‘Ca. P. solani’, have broad plant host range and are transmitted by several polyphagous insect species. To understand better how these obligate symbionts can colonize such a wide range of hosts, the genome of ‘Ca. P. solani’ strain SA-1 was sequenced from infected periwinkle via a metagenomics approach. The de novo assembly generated a draft genome with 19 contigs totalling 821,322 bp, which corresponded to more than 80% of the estimated genome size. Further completion of the genome was challenging due to the high occurrence of repetitive sequences. The majority of repeats consisted of gene arrangements characteristic of phytoplasma potential mobile units (PMUs). These regions showed variation in gene orders intermixed with genes of unknown functions and lack of similarity to other phytoplasma genes, suggesting that they were prone to rearrangements and acquisition of new sequences via recombination. The availability of this high-quality draft genome also provided a foundation for genome-scale genotypic analysis (e.g., average nucleotide identity and average amino acid identity) and molecular phylogenetic analysis. Phylogenetic analyses provided evidence of horizontal transfer for PMU-like elements from various phytoplasmas, including distantly related ones. The ‘Ca. P. solani’ SA-1 genome also contained putative secreted protein/effector genes, including a homologue of SAP11, found in many other phytoplasma species.