Post-penetration Stages Research Articles

Anti-DENV-2 Activity of Ethanolic Extracts from Arachis hypogaea L.: Peanut Skin as a Relevant Resource of Bioactive Compounds against Dengue Virus

Dengue is an emerging disease of high impact on human health. Plants are an important source of new antivirals and Arachis hypogaea stands for its biological properties. The aim of this study was to evaluate the cytotoxicity and antiviral activity and elucidate the antiviral mechanism of ethanolic extracts from A. hypogaea against dengue virus 2 (DENV-2). The skin or tegument ethanolic extract (TEEs) and seed ethanolic extract (SEEs) were obtained. Cytotoxicity was evaluated by MTT and Neutral Red Uptake (NRU). Antiviral activity was evaluated at different stages of the viral replication cycle by the lysis plaque reduction method. The 50% inhibitory concentration (IC50) and selectivity index (SI) were determined. Antiviral activity was further determined by RT-qPCR. The CC50 values were 169 (NRU) and 65 (MTT) µg/mL for TEE. In addition, the CC50 values were >1400 (NRU) and 636 (MTT) µg/mL for SEE. The TEE demonstrated 99.9 ± 0.1% viral inhibition. The TEE presented an IC50 = 3.47 and SI of 48.7 (NRU) and 18.73 (MTT). Its mechanism of antiviral action is broad and it acts in the viral adsorption–penetration stage and inhibits the first steps of infection in the post-penetration stage. It is also capable of acting as virucidal and as prophylactic. Studies of RT-qPCR indicated that the TEE inhibited viral RNA synthesis. These findings suggest that the TEE from A. hypogaea could be a promising antiviral candidate for treating DENV-2 infections.

Cacao pod transcriptome profiling of seven genotypes identifies features associated with post-penetration resistance to Phytophthora palmivora

The oomycete Phytophthora palmivora infects the fruit of cacao trees (Theobroma cacao) causing black pod rot and reducing yields. Cacao genotypes vary in their resistance levels to P. palmivora, yet our understanding of how cacao fruit respond to the pathogen at the molecular level during disease establishment is limited. To address this issue, disease development and RNA-Seq studies were conducted on pods of seven cacao genotypes (ICS1, WFT, Gu133, Spa9, CCN51, Sca6 and Pound7) to better understand their reactions to the post-penetration stage of P. palmivora infection. The pod tissue-P. palmivora pathogen assay resulted in the genotypes being classified as susceptible (ICS1, WFT, Gu133 and Spa9) or resistant (CCN51, Sca6 and Pound7). The number of differentially expressed genes (DEGs) ranged from 1625 to 6957 depending on genotype. A custom gene correlation approach identified 34 correlation groups. De novo motif analysis was conducted on upstream promoter sequences of differentially expressed genes, identifying 76 novel motifs, 31 of which were over-represented in the upstream sequences of correlation groups and associated with gene ontology terms related to oxidative stress response, defense against fungal pathogens, general metabolism and cell function. Genes in one correlation group (Group 6) were strongly induced in all genotypes and enriched in genes annotated with defense-responsive terms. Expression pattern profiling revealed that genes in Group 6 were induced to higher levels in the resistant genotypes. An additional analysis allowed the identification of 17 candidate cis-regulatory modules likely to be involved in cacao defense against P. palmivora. This study is a comprehensive exploration of the cacao pod transcriptional response to P. palmivora spread after infection. We identified cacao genes, promoter motifs, and promoter motif combinations associated with post-penetration resistance to P. palmivora in cacao pods and provide this information as a resource to support future and ongoing efforts to breed P. palmivora-resistant cacao.

Analysis of Asian Rice (Oryza sativa) Genotypes Reveals a New Source of Resistance to the Root-Knot Nematode Meloidogyne javanica and the Root-Lesion Nematode Pratylenchus zeae

The sedentary root-knot nematodes, Meloidogyne spp., and the migratory root-lesion nematodes, Pratylenchus spp., cause significant yield losses, particularly in aerobic and upland rice production systems. Recently, the Asian rice Oryza sativa accessions LD 24 and Khao Pahk Maw (KPM) were shown to be highly resistant to M. graminicola. In this study, we have analyzed the responses and broadness of resistance of these two rice genotypes to another root-knot nematode M. javanica and a root-lesion nematode P. zeae. The penetration as well as post-penetration development and reproduction of nematodes were compared including known susceptible and resistant genotypes. Our results indicate that the genotype KPM confers strong resistance to both M. javanica and P. zeae, while LD 24 was resistant to M. javanica and susceptible to P. zeae. Detailed observations revealed that similar numbers of M. javanica or P. zeae penetrated the resistant and susceptible hosts during early infection stages. However, the development and reproduction of both nematodes were arrested or reduced in resistant genotypes, implying that resistance occurs at the post-penetration stage.

Deficiencies in the Mitochondrial Electron Transport Chain Affect Redox Poise and Resistance Toward Colletotrichum higginsianum.

To investigate if and how the integrity of the mitochondrial electron transport chain (mETC) influences susceptibility of Arabidopsis toward Colletotrichum higginsianum, we have selected previously characterized mutants with defects at different stages of the mETC, namely, the complex I mutant ndufs4, the complex II mutant sdh2-1, the complex III mutant ucr8-1, and a mutant of the uncoupling protein ucp1-2. Relative to wild type, the selected complex I, II, and III mutants showed decreased total respiration, increased alternative respiration, as well as increased redox charge of the NADP(H) pool and decreased redox charge of the NAD(H) pool in the dark. In the light, mETC mutants accumulated free amino acids, albeit to varying degrees. Glycine and serine, which are involved in carbon recycling from photorespiration, and N-rich amino acids were predominantly increased in mETC mutants compared to the wild type. Taking together the physiological phenotypes of all examined mutants, our results suggest a connection between the limitation in the re-oxidation of reducing equivalents in the mitochondrial matrix and the induction of nitrate assimilation into free amino acids in the cytosol, which seems to be engaged as an additional sink for reducing power. The sdh2-1 mutant was less susceptible to C. higginsianum and did not show hampered salicylic acid (SA) accumulation as previously reported for SDH1 knock-down plants. The ROS burst remained unaffected in sdh2-1, emonstrating that subunit SDH2 is not involved in the control of ROS production and SA signaling by complex II. Moreover, the ndufs4 mutant showed only 20% of C. higginsianum colonization compared to wild type, with the ROS burst and the production of callose papillae being significantly increased compared to wild type. This indicates that a restriction of respiratory metabolism can positively affect pre-penetration resistance of Arabidopsis. Taking metabolite profiling data from all investigated mETC mutants, a strong positive correlation of resistance toward C. higginsianum with NADPH pool size, pyruvate contents, and other metabolites associated with redox poise and energy charge was evident, which fosters the hypothesis that limitations in the mETC can support resistance at post-penetration stages by improving the availability of metabolic power.

Transcriptome analysis reveals pathways facilitating the growth of tobacco powdery mildew in Arabidopsis

Powdery mildew (PM) fungi are biotrophic pathogens that rely on living hosts to survive and thrive. However, their colonization is restricted by host defenses at both the penetration and post-penetration stages. The tobacco PM strain Golovinomyces cichoracearum (Gc) SICAU1 has overcome penetration resistance of Arabidopsis but its growth is arrested by post-penetration resistance. While Gc SICAU1 only poorly grows in Arabidopsis Col-0 wild-type plants, it can sustainably grow for more than 20 days on the same infected leaves of the double mutant pad4–1 sid2–1 that is defective in both the synthesis and signaling of salicylic acid (SA). To understand the underlying molecular mechanisms, we conducted a comparative transcriptome analysis between Col-0 and pad4–1 sid2–1 in response to Gc SICAU1. We found that 4811 genes were differentially expressed more than four-fold between any two of the measured seven time points (0, 1, 3, 6, 8, 10 and 12 days post-inoculation). Gene expression pattern analysis suggests that differential expression of 348 genes and 190 genes may explain resistance in Col-0 and susceptibility in pad4–1 sid2–1, respectively. Gene Ontology (GO) analysis suggests that Gc SICAU1 might be arrested in Col-0 by both pattern-triggered immunity and SA-dependent defense. By contrast, its sustained growth in pad4–1 sid2–1 may be attributable to the activation of a detoxification pathway that is normally repressed by the SA-signaling pathway. Taken together, our results suggest that multiple distinct, yet interconnected pathways control the growth of tobacco powdery mildew in Arabidopsis.

Embryogenesis, penetration and post penetration development of Meloidogyne enterolobii in Guava (Psidium guajava L.)

Guava (Psidium guajava L.) is one of the important commercial fruit crops grown in India. A new species of rootknot nematode Meloidogyne enterolobii reported first from India by Poornima et al., 2016, has become a fast spreading problem in guava cultivation causing severe reduction in yield and decline of guava orchards across the country and world. The present study was taken up to observe the activity of developmental stages such as embryonic, penetration and post penetration stages of M. enterolobii in order to get a clear picture of the vulnerable stage for managing the nematode. It was observed that the average size of a viable egg was 42.48 μm (±4.20 μm) in length and 20.21 μm (±1.70 μm) in width (n = 10). The average number of eggs/egg masses was 179 (±16) (n = 10). These dimensions are comparable to the dimensions described for eggs of many other plant parasitic nematode species. The 3rd stage juveniles were observed in 2–3 days after Jstage and Jstages were observed in 4–5 days after J3 stage. The swollen female or immature stage of females were seen at 2–3 days after J4 stage, fully developed female was observed in guava roots at 24–28 days starting with single cell stage, the female starts laying eggs at 28–30 days from single cell stage. The optimal temperature for development of M. enterolobii is 28°C, and corresponds to the geographical distribution of this nematode in subtropical regions. At this temperature it takes approximately 3 weeks to complete one life cycle.

Elucidation of the functional role of flagella in virulence and ecological traits of Pseudomonas cichorii using flagella absence (ΔfliJ) and deficiency (ΔfliI) mutants

Understanding the infection mechanisms of pathogens will lead to better management of the associated diseases. The flagella of these pathogens play significant roles not only in bacterial motility, but also in virulence. In the present study, two genes involved in flagella construction, fliJ and fliI of Pseudomonas cichorii, were analyzed. The results revealed that these genes are vital for flagella formation and play significant roles not only in motility, but also in virulence. When we inoculated host plants with fliI- and fliJ-defective mutants (ΔfliJ and ΔfliI) through the dipping method, the degree of disease severity caused by both mutants was significantly reduced compared to those of the wild-type. However, the virulence of ΔfliI was stronger than that of ΔfliJ. Electron microscope observation, and swarming and leaf attachment assays indicated a reduced number of flagella in ΔfliI, but not complete absence, because of the presence of another copy of fliI. Furthermore, a vacuum infiltration assay revealed that flagella are indispensable in the pre- and post-penetration stages for complete virulence. Overall, we created semi-defective (ΔfliI) and completely defective (ΔfliJ) mutants and elucidated the fact that flagella play significant roles in virulence of the pathogen at different stages of the infection process.

Histochemical Characterization of Early Response to Cochliobolus sativus Infection in Selected Barley Genotypes

Much effort is being made to breed barley with durable resistance to leaf spot blotch incited by Bipolaris sorokiniana (teleomorph: Cochliobolus sativus). We hypothesized that susceptibility and resistance traits in 11 diverse barley genotypes inoculated with a single C. sativus isolate might specify a range of distinct host cell responses. Quantitative descriptions of interaction microphenotypes exhibited by different barley genotype seedlings after infection with C. sativus are provided. Early oxidative responses occurring in epidermis and mesophyll leaf tissue were monitored by histochemical analysis of H2O2 accumulation at 8, 24, and 48 h after inoculation. Cell wall apposition (CWA) in epidermal cells and hypersensitive reaction (HR) of epidermal or mesophyll tissue were early defenses in both resistant and susceptible genotypes. There were differences in level, duration, and frequency of occurrence for CWA and HR for the different barley genotypes. Occurrence of HR in epidermal cells at post-penetration stages was indicative of compatibility. Patterns of cell responses were microphenotypically diverse between different resistant and susceptible genotypes. This suggests that timing and level of response are key features of microphenotypic diversity that distinguish different functional mechanisms of resistance and susceptibility present in barley.

Resistance to powdery mildew in one Spanish barley landrace hardly resembles other previously identified wild barley resistances

Two major quantitative trait loci (QTLs) associated with resistance to powdery mildew (Blumeria graminis f. sp. hordei) were previously identified on chromosome 7H of the Spanish barley line SBCC097. The two QTLs seemed to share the same chromosomal position as the major genes mlt and Mlf, which were formerly described in Hordeum vulgare ssp. spontaneum-derived lines. In the present work, different lines that carry mlt (RS42-6*O), Mlf (RS137-28*E), or a combination of both (SI-4 and SI-6) were compared with SBCC097 to evaluate their relatedness at the phenotypic, cellular, and genetic levels. The resistance of the lines was characterised by inoculating them with a set of 27 isolates of B. graminis, which displayed a wide range of virulence. It was revealed that SBCC097 possessed a distinctive resistance spectrum. Microscopic assessment of the cytological development of the resistance response showed that SBCC097 clearly formed fewer well-established colonies and secondary hyphae than the other lines. This was confirmed by the infection type recorded after visual inspection. Genetic analyses of all five lines, based on markers flanking the QTLs derived from SBCC097, supported the macroscopic and microscopic data and pointed to the presence of a combination of novel genes or alleles in SBCC097, which may be included in the category of “intermediate-acting” genes, governing resistance mainly at the post-penetration stage.

Common Motifs in the Response of Cereal Primary Metabolism to Fungal Pathogens are not Based on Similar Transcriptional Reprogramming.

During compatible interactions with their host plants, biotrophic plant–pathogens subvert host metabolism to ensure the sustained provision of nutrient assimilates by the colonized host cells. To investigate, whether common motifs can be revealed in the response of primary carbon and nitrogen metabolism toward colonization with biotrophic fungi in cereal leaves, we have conducted a combined metabolome and transcriptome study of three quite divergent pathosystems, the barley powdery mildew fungus (Blumeria graminis f.sp. hordei), the corn smut fungus Ustilago maydis, and the maize anthracnose fungus Colletotrichum graminicola, the latter being a hemibiotroph that only exhibits an initial biotrophic phase during its establishment. Based on the analysis of 42 water-soluble metabolites, we were able to separate early biotrophic from late biotrophic interactions by hierarchical cluster analysis and principal component analysis, irrespective of the plant host. Interestingly, the corresponding transcriptome dataset could not discriminate between these stages of biotrophy, irrespective, of whether transcript data for genes of central metabolism or the entire transcriptome dataset was used. Strong differences in the transcriptional regulation of photosynthesis, glycolysis, the TCA cycle, lipid biosynthesis, and cell wall metabolism were observed between the pathosystems. However, increased contents of Gln, Asn, and glucose as well as diminished contents of PEP and 3-PGA were common to early post-penetration stages of all interactions. On the transcriptional level, genes of the TCA cycle, nucleotide energy metabolism and amino acid biosynthesis exhibited consistent trends among the compared biotrophic interactions, identifying the requirement for metabolic energy and the rearrangement of amino acid pools as common transcriptional motifs during early biotrophy. Both metabolome and transcript data were employed to generate models of leaf primary metabolism during early biotrophy for the three investigated interactions.

The Ustilago maydis Forkhead Transcription Factor Fox1 Is Involved in the Regulation of Genes Required for the Attenuation of Plant Defenses During Pathogenic Development

Ustilago maydis is a plant-pathogenic fungus that establishes a biotrophic relationship with its host plant, Zea mays. The pathogenic stage of U. maydis is initiated by the fusion of two haploid cells, resulting in the formation of a dikaryotic hypha that invades the plant cell. The switch from saprophytic, yeast-like cells to the biotrophic hyphae requires the complex regulation of a multitude of biological processes to constitute the compatible host-fungus interaction. Transcriptional regulators involved in the establishment of the infectious dikaryon and penetration of the host tissue have been identified; however, regulators required during the post-penetration stages remained to be elucidated. In this study, we report the identification of a U. maydis forkhead transcription factor, Fox1, which is exclusively expressed during biotrophic development. Deletion of fox1 results in reduced virulence and impaired tumor development. The Deltafox1 hyphae induce the accumulation of H(2)O(2) in and around infected cells and a maize defense response phenotypically represented by the encasement of proliferating hyphae in a cellulose-containing matrix. The phenotype can be attributed to the fox1-dependent deregulation of several effector genes that are linked to pathogenic development and host defense suppression.

Efficacy of screening methods used in breeding for black pod disease resistance varieties in cocoa

Black pod is an important fungal disease of cocoa (Theobroma cacao L.) that causes severe yield losses. The disease affects the pods, beans, flower cushions, leaves, stems and roots. The objective of this study was to evaluate the efficacy of resistance screening methods used in breeding black pod disease resistance in cocoa. The resistance of cocoa leaves and pods to Phytophthora palmivora was investigated in twenty five international genotypes of cocoa in five laboratory experiments and a field observation at the Cocoa Research Institute ofGhana. A significant clonal difference for leaf and pod resistance was observed at penetration and post-penetration stages of infection. Correlation between resistance of leaves and pods was positive and significant at both penetration and post-penetration stages of infection. The occurrence of such a characteristic of cocoa leaf suggests the value of use of leaves of cocoa seedlings for the prediction of pod resistance to black pod disease. The reliability of the tests was evaluated by correlating results of the inoculation tests with the level of field infection. These correlations were generally positive and significant, both for detached pod and leaf tests. The high positive correlation between detached leaves and pods and natural Phytophthora pod rot infection in the field showed that detached organs can be used for prediction of resistance in the field. Overall, for the convenience ofoperations and reliability of results, leaf disc test was the best screening method and was also time saving. Clones SCA 6, T85/799, LAF1, ICS 1 and GU 225V were noted to be promising for breeding against black pod disease. The susceptible clones were MO 20, T79/501, VENC 4-4, PA 120 and MOCORONGO.

Molecular Cloning and Characterization of a Chitinase-Homologous Gene from Mikania micrantha Infected by Cuscuta campestris

Mmchi1, a putative chitinase gene from Mikania micrantha infected by the parasitic plant Cuscuta campestris, was cloned and characterized. Mmchi1 is predicted to encode a 35.42-kDa polypeptide with a isoelectric point of 5.69. The corresponding genomic sequence contains two introns (1,171 and 621 bp). Phylogenetic analysis showed that the predicted Mmchi1 protein is related to class I and class II chitinases (glycoside hydrolase family 19). Mmchi1 is likely a class II chitinase, as the protein sequence lacks a cysteine-rich hevein domain at its N terminus. Southern blot analysis suggests that sequences related to Mmchi1 exist in the genome of M. micrantha. Expression of Mmchi1 in different tissues was analyzed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Mmchi1 was constitutively expressed in shoots, whereas only low transcript levels were detected in other tissues. Transcript levels of Mmchi1 in shoots of M. micrantha infected by C. campestris, as analyzed by RT-PCR and real-time PCR, significantly increased after 2 days post infection but markedly decreased during the following days. Transcript levels of Mmchi1 determined by semiquantitative RT-PCR varied in noninfected shoots exposed to various stress factors. Elevated levels of Mmchi1 transcripts were accumulated in response to mechanical wounding and application of abscisic acid, salicylic acid, or ZnSO4, respectively. Under salt stress conditions, Mmchi1 transcripts accumulated at significant lower levels, however. Taken together, these data suggest that Mmchi1 is a stress-related gene of M. micrantha, which is stimulated in response to C. campestris infection at early post-penetration stage but significantly suppressed during later infection stages.

Identification of genes differentially expressed in Mikania micrantha during Cuscuta campestris infection by suppression subtractive hybridization

The influence of Cuscuta campestris on its host Mikania micrantha has been studied with respect to biomass accumulation, physiology and ecology. Molecular events of this parasitic plant-plant interaction are poorly understood, however. In this study, we identified novel genes from M. micrantha induced by C. campestris infection. Genes expressed upon parasitization by C. campestris at early post-penetration stages were investigated by construction and characterization of subtracted cDNA libraries from shoots and stems of M. micrantha. Three hundred and three presumably up-regulated expressed sequence tags (ESTs) were identified and classified in functional categories, such as "metabolism", "cell defence and stress", "transcription factor", "signal transduction", "transportation" and "photosynthesis". In shoots and stems of infected M. micrantha, genes associated with defence responses and cell wall modifications were induced, confirming similar data from other parasitic plant-plant interactions. However, gene expression profiles in infected shoots and stems were found to be different. Compared to infected shoots, more genes induced in response to biotic and abiotic stress factors were identified in infected stems. Furthermore, database comparisons revealed a notable number of M. micrantha ESTs that matched genes with unknown function. Expression analysis by quantitative real-time RT-PCR of 21 genes (from different functional categories) showed significantly increased levels for 13 transcripts in response to C. campestris infection. In conclusion, this study provides an overview of genes from parasitized M. micrantha at early post-penetration stages. The acquired data form the basis for a molecular understanding of host reactions in response to parasitic plants.

Proteasomal Degradation of TRIM5α during Retrovirus Restriction

The host protein TRIM5α inhibits retroviral infection at an early post-penetration stage by targeting the incoming viral capsid. While the detailed mechanism of restriction remains unclear, recent studies have implicated the activity of cellular proteasomes in the restriction of retroviral reverse transcription imposed by TRIM5α. Here, we show that TRIM5α is rapidly degraded upon encounter of a restriction-susceptible retroviral core. Inoculation of TRIM5α-expressing human 293T cells with a saturating level of HIV-1 particles resulted in accelerated degradation of the HIV-1-restrictive rhesus macaque TRIM5α protein but not the nonrestrictive human TRIM5α protein. Exposure of cells to HIV-1 also destabilized the owl monkey restriction factor TRIMCyp; this was prevented by addition of the inhibitor cyclosporin A and was not observed with an HIV-1 virus containing a mutation in the capsid protein that relieves restriction by TRIMCyp IVHIV. Likewise, human TRIM5α was rapidly degraded upon encounter of the restriction-sensitive N-tropic murine leukemia virus (N-MLV) but not the unrestricted B-MLV. Pretreatment of cells with proteasome inhibitors prevented the HIV-1-induced loss of both rhesus macaque TRIM5α and TRIMCyp proteins. We also detected degradation of endogenous TRIM5α in rhesus macaque cells following HIV-1 infection. We conclude that engagement of a restriction-sensitive retrovirus core results in TRIM5α degradation by a proteasome-dependent mechanism.

Transcript Profiles of Blumeria graminis Development During Infection Reveal a Cluster of Genes That Are Potential Virulence Determinants

High-density cDNA microarrays (2,027 unigenes) were used to analyze transcript profiles of the plant-pathogenic fungus Blumeria graminis f. sp. hordei throughout its asexual life cycle and development of infection. RNA was obtained from four stages preceding penetration and four stages after penetration of the host cells. The microarray data was validated by comparing the expression of a plasma membrane H+-ATPase and fructose-1,6-bis phosphatase with the data obtained from a quantitative polymerase chain reaction (PCR) assay. The results showed that there was a global switch in expression between the pre- and postpenetrative stages. This was largely due to accumulation of RNA encoding protein biosynthesis genes in the late stages. Other functional clusters, such as virulence-related genes and sterol metabolism genes, are up-regulated in pre- and postpenetration stages, respectively. A group of RNAs whose abundance correlated with the expression of cap20, a gene known to be required for virulence in Colletotrichum gloeosporioides, identified genes that are strong candidates for pathogenicity factors in B. graminis.

Cellular and viral specificities of human immunodeficiency virus type 1 vif protein.

The vif gene of human immunodeficiency virus type 1 (HIV-1) greatly enhances the infectivity of HIV-1 virions that are released from cells classified as nonpermissive (e.g., lymphocytes, macrophages, and H9 leukemic T cells) but is irrelevant in permissive cells (e.g., HeLa or COS cells). Recently, it was reported that vif expression in nonpermissive cells dramatically increases infectivity not only of HIV-1 but also of other enveloped viruses, including murine leukemia viruses (MLVs). This was surprising in part because MLVs and other murine retroviruses lack vif genes yet replicate efficiently in T lymphocytes. To investigate these issues, we first developed improved methods for producing substantial quantities of HIV-1 virions with vif deletions from healthy H9 cells. These virions had approximately the same amounts of major core proteins and envelope glycoproteins as the control wild-type virions but were only approximately 1% as infectious. We then produced H9 cells that contained wild-type or vif deletion HIV-gpt proviruses, which lack a functional env gene. After superinfection with either xenotropic or amphotropic MLVs, these cells released HIV-gpt virions pseudotyped with an MLV envelope plus replication-competent MLV. Interestingly, the pseudotyped HIV-gpt (vif deletion) virions were noninfectious, whereas the MLV virions simultaneously released from the same H9 cells were fully infectious. These results strongly suggest that the Vif protein functions in a manner that is both cell specific and at least substantially specific for HIV-1 and related lentiviruses. In addition, these results confirm that vif deletion HIV-1 virions from nonpermissive cells are blocked at a postpenetration stage of the infection pathway.

Foliar Resistance to Phytophthora palmivora as an Indicator of Pod Resistance in Theobroma cacao

Resistance to Phytophthora palmivora was assessed in leaves and pods of 12 diverse cacao genotypes at the penetration and post-penetration stages of infection to determine the relationship of resistance between stages and sites of infection. Penetration resistance was based on the frequency of lesions, whereas lesion size was used as a measure of post-penetration resistance. We observed significant clonal differences for leaf and pod resistance at the two stages of infection. A poor correlation between resistance at the penetration and post-penetration stages in both leaves and pods indicated that these two forms of resistance are independent and governed by different mechanisms. Correlation between resistance of leaves and pods was not significant at the penetration stage, whereas that of post-penetration resistance was positive and significant. This shows that internal or tissue resistance is common between leaf and pod and indicates that leaf resistance at the post-penetration stage of infection could be used to predict pod resistance; however, resistance of leaves at the penetration stage cannot be used to predict penetration resistance in pods. A high positive correlation obtained between attached leaves and pods with their detached counterparts showed that detached organs can be used for the prediction of resistance in attached leaves and pods.

Inheritance of foliar resistance to Phytophthora palmivora (Butler) Butler in cacao (Theobroma cacao L.)

The genetic nature of resistance in leaves of cacao (Theobroma cacao L.) to Phytophthora palmivora (Butler) Butler was investigated in a 3 × 8 and a 4 × 4 mating design. Leaf resistance was assessed at the penetration and post-penetration stages of infection. Wide variability was observed among the 32 families examined for lesion frequency (penetration resistance) and lesion size / spread (post-penetration resistance). Segregation within families showed a continuous distribution for both forms of resistance, suggesting that resistance is polygenic. The poor association between resistance at the penetration stage and that at the post-penetration stage suggested that they were independently inherited and could be selected independently with the aim of accumulating genes for resistance. Genetic complementation between the two forms of resistance and transgressive segregation were evident within the families studied. The additive effects were predominant for lesion size, while both additive and non-additive effects were important in the inheritance of lesion frequency. The narrow sense heritabilities obtained from North Carolina Model II analysis were as high as 0.61 and 0.40 for lesion size and lesion frequency respectively. Possible breeding strategies based on these findings are discussed.

Light and scanning electron microscope studies of leaf blotch of onion caused byCladosporium allii–cepae

The pre–penetration and post–penetration stages of infection byCladosporium allii–cepaeon onion foliage was examined on inoculated plants kept at 15° C and 80–90% r.h. The pathogen entered the leaf usually through stomata but occasionally by penetrating the cuticle. Invasion of the palisade and mesophyll tissues led to the formation of a leaf cavity after 7 days and after 30 days the pathogen sporulated on the leaf surface.