Moniliophthora roreri is the fungal pathogen that causes frosty pod rot (FPR) disease of Theobrom... more Moniliophthora roreri is the fungal pathogen that causes frosty pod rot (FPR) disease of Theobroma cacao L., the source of chocolate. FPR occurs in most of the cacao producing countries in the Western Hemisphere, causing yield losses up to 80%. Genetic diversity within the FPR pathogen population may allow the population to adapt to changing environmental conditions and adapt to enhanced resistance in the host plant. The present study developed single nucleotide polymorphism (SNP) markers from RNASeq results for 13 M. roreri isolates and validated the markers for their ability to reveal genetic diversity in an international M. roreri collection. The SNP resources reported herein represent the first study of RNA sequencing (RNASeq)-derived SNP validation in M. roreri and demonstrates the utility of RNASeq as an approach for de novo SNP identification in M. roreri. A total of 88 polymorphic SNPs were used to evaluate the genetic diversity of 172 M. roreri cacao isolates resulting in 37 distinct genotypes (including 14 synonymous groups). Absence of heterozygosity for the 88 SNP markers indicates reproduction in M. roreri is clonal and likely due to a homothallic life style. The upper Magdalena Valley of Colombia showed the highest levels of genetic diversity with 20 distinct genotypes of which 13 were limited to this region, and indicates this region as the possible center of origin for M. roreri.
Theobroma cacao pods were inoculated with meiospores of Moniliophthora roreri (Mr), a hemibiotrop... more Theobroma cacao pods were inoculated with meiospores of Moniliophthora roreri (Mr), a hemibiotrophic basidiomycete causing frosty pod rot. Pods were malformed 30 days after inoculation (DAI) and sporulation was observed 60 DAI. Glucose and asparagine concentrations decreased and mannitol and malonate increased in infected pods 30 DAI. By 60 DAI, most carbohydrates, amino acids, and organic acids were drastically reduced by infection. Mannitol and succinic acid levels increased 60 DAI and likely originated from Mr. RT-qPCR analysis of cacao ESTs indicated a strong response to infection 30 DAI in malformed pod. Evidence indicated that biotrophic hyphae colonized pods and a shift to necrotrophic growth occurred later (during the end stages of infection). Expression of cacao ESTs associated with plant hormone biosynthesis and action was altered. Changes in the expression of Mr ESTs in response to nutrient deficiency in pure culture were small. Changes in Mr gene expression patterns and levels of specific metabolites in necrotic sporulating pods 60 DAI compared to malformed pods 30 DAI indicated that the glyoxylate cycle of Mr was up regulated during the shift from biotrophic to necrotrophic phases of the disease cycle.
Drought can negatively impact pod production despite the fact that cacao production usually occur... more Drought can negatively impact pod production despite the fact that cacao production usually occurs in tropical areas having high rainfall. Polyamines (PAs) have been associated with the response of plants to drought in addition to their roles in responses to many other stresses. The constitutive and drought inducible expression patterns of genes encoding enzymes involved in PA biosynthesis were determined:
Endophytic isolates of Trichoderma species are being considered as biocontrol agents for diseases... more Endophytic isolates of Trichoderma species are being considered as biocontrol agents for diseases of Theobroma cacao (cacao). Gene expression was studied during the interaction between cacao seedlings and four endophytic Trichoderma isolates, T. ovalisporum-DIS 70a, T. hamatum-DIS 219b, T. harzianum-DIS 219f, and Trichoderma sp.-DIS 172ai. Isolates DIS 70a, DIS 219b, and DIS 219f were mycoparasitic on the pathogen Moniliophthora roreri, and DIS 172ai produced metabolites that inhibited growth of M. roreri in culture. ESTs (116) responsive to endophytic colonization of cacao were identified using differential display and their expression analyzed using macroarrays. Nineteen cacao ESTs and 17 Trichoderma ESTs were chosen for real-time quantitative PCR analysis. Seven cacao ESTs were induced during colonization by the Trichoderma isolates. These included putative genes for ornithine decarboxylase (P1), GST-like proteins (P4), zinc finger protein (P13), wound-induced protein (P26), EF-calcium-binding protein (P29), carbohydrate oxidase (P59), and an unknown protein (U4). Two plant ESTs, extensin-like protein (P12) and major intrinsic protein (P31), were repressed due to colonization. The plant gene expression profile was dependent on the Trichoderma isolate colonizing the cacao seedling. The fungal ESTs induced in colonized cacao seedlings also varied with the Trichoderma isolate used. The most highly induced fungal ESTs were putative glucosyl hydrolase family 2 (F3), glucosyl hydrolase family 7 (F7), serine protease (F11), and alcohol oxidase (F19). The pattern of altered gene expression suggests a complex system of genetic cross talk occurs between the cacao tree and Trichoderma isolates during the establishment of the endophytic association.
Yield of Theobroma cacao (cacao), the source of chocolate, is limited by disease and insect pests... more Yield of Theobroma cacao (cacao), the source of chocolate, is limited by disease and insect pests. Developmental stage influences the resistance of cacao leaves to disease and insect pests. Mechanical wounding, ethylene, and methyl jasmonate induce resistance to pests in many plant species. The effects of mechanical wounding, ethylene, and methyl jasmonate on gene expression was studied in cacao leaves at two developmental stages, young red (YR) and mature green (MG). Differential expression was observed for genes putatively encoding a DNA binding protein (TcWRKY-1), a protein regulating cell division (TcORFX-1), a Type III peroxidase (TcPer-1), an endo-1,4-b-glucanase (TcGlu-1), a class VII chitinase (TcChiB), a caffeine synthase (TcCaf-1), and a light-harvesting complex protein (TcLhca-1). Wounding induced TcWRKY-1 and TcORFX-1 in YR and MG leaves. Elevated TcPer-1 mRNA levels were detected in YR and MG leaves after wounding. Wounding induced TcChiB in YR leaves and repressed TcLhca-1 in MG leaves. Ethylene (12 mL L À1 ) induced TcPer-1 in YR and MG leaves but induced TcGlu-1 in MG leaves only. Ethylene repressed TcLhca-1 and TcCaf-1 in YR leaves. Ethylene repressed TcLhca-1 and TcChiB in MG leaves. Methyl jasmonate (0.2 mM) induced TcCaf-1 and TcChiB in YR leaves and TcPer-1 and TcChiB in MG leaves. Ethylene/methyl jasmonate combined induced TcChiB in YR leaves and TcGlu-1 in MG leaves. 1-Methylcyclopropene, an inhibitor of ethylene action, blocked ethylene induced responses but did not block responses to wounding or methyl jasmonate. The cacao response to wounding, ethylene, and/or methyl jasmonate was influenced by developmental stage. Cross-talk between ethylene and methyl jasmonate action on cacao gene expression resulted in synergistic and antagonistic responses. It is critical to account for tissue developmental stage when studying the molecular responses of cacao to mechanical wounding, ethylene, and methyl jasmonate. The constituative and inducible defense strategies used by cacao are dependent on the developmental stage of the tissues involved. #
Developmental expression of stress response genes in Theobroma cacao leaves and their response to... more Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 and a compatible infection by Phytophthora megakarya were studied. Ten genes were selected to represent genes involved in defense TcGlu1, TcChiB,, gene regulation (TcWRKY-1 and TcORFX-1), cell wall development (TcCou-1, TcPer-1, and TcGlu-1), or energy production (TcLhca-1 and TcrbcS). Leaf development was separated into unexpanded (UE), young red (YR), immature green (IG), and mature green (MG). Our data indicates that the constitutive defense mechanisms used by cacao leaves differ between different developmental stages. TcWRKY-1 and TcChiB were highly expressed in MG leaves, and TcPer-1, TcGlu-1, and TcCou-1 were highly expressed inYR leaves. TcGlu1,3 was highly expressed in UE and YR leaves, TcCaf-1 was highly expressed in UE leaves, and TcLhca-1 and TcrbcS were highly expressed in IG and MG leaves. NEP1 encodes the necrosis inducing protein Nep1 produced by Fusarium oxysporum and has orthologs in Phytophthora species. Nep1 caused cellular necrosis on MG leaves and young pods within 24 h of application. Necrosis was observed on YR leaves 10 days after treatment. Expression of TcWRKY-1, TcORFX-1, TcPer-1, and TcGlu-1 was enhanced and TcLhca-1 and TcrbcS were repressed in MG leaves after Nep1 treatment. Expression of TcWRKY-1 and TcORFX-1 was enhanced in YR leaves after Nep1 treatment. Infection of MG leaf disks by P. megakarya zoospores enhanced expression of TcGlu-1, TcWRKY-1, and TcPer-1 and repressed expression of TcChiB, TcLhca-1 and TcrbcS. Five of the six genes that were responsive to Nep1 were responsive to infection by P. megakarya. Susceptibility of T. cacao to P. megakarya includes altered plant gene expression and phytotoxic molecules like Nep1 may contribute to susceptibility. Published by Elsevier SAS.
Drought can negatively impact pod production despite the fact that cacao production usually occur... more Drought can negatively impact pod production despite the fact that cacao production usually occurs in tropical areas having high rainfall. Polyamines (PAs) have been associated with the response of plants to drought in addition to their roles in responses to many other stresses. The constitutive and drought inducible expression patterns of genes encoding enzymes involved in PA biosynthesis were determined: an ornithine decarboxylase (TcODC ), an arginine decarboxylase (TcADC ), an S-adenosylmethionine decarboxylase (TcSAMDC ), a spermidine synthase (TcSPDS ), and a spermine synthase (TcSPMS ). Expression analysis using quantitative real-time reverse transcription-PCR (QPCR) results showed that the PA biosynthesis genes were expressed in all plant tissues examined. Constitutive expression of PA biosynthesis genes was generally highest in mature leaves and open flowers. Expression of TcODC, TcADC, and TcSAMDC was induced with the onset of drought and correlated with changes in stomatal conductance, photosynthesis, photosystem II efficiency, leaf water potential and altered emission of blue-green fluorescence from cacao leaves. Induction of TcSAMDC in leaves was most closely correlated with changes in water potential. The earliest measured responses to drought were enhanced expression of TcADC and TcSAMDC in roots along with decreases in stomatal conductance, photosynthesis, and photosystem II efficiency. Elevated levels of putrescine, spermidine, and spermine were detected in cacao leaves 13 days after the onset of drought. Expression of all five PA associated transcripts was enhanced (1.5e3-fold) in response to treatment with abscisic acid. TcODC and TcADC, were also responsive to mechanical wounding, infection by Phytophthora megakarya (a causal agent of black pod disease in cacao), the necrosis-and ethylene-inducing protein (Nep1) of Fusarium oxysporum, and flower abscission. TcSAMDC expression was responsive to all stresses except flower abscission. TcODC, although constitutively expressed at much lower levels than TcADC, TcSAMDC, TcSPDS, and TcSPMS, was highly inducible by the fungal protein Nep1 (135-fold) and the cacao pathogen Phytophthora megakarya (671-fold). The full length cDNA for ODC was cloned and characterized. Among the genes studied, TcODC, TcADC, and TcSAMDC were most sensitive to induction by drought in addition to other abiotic and biotic stresses. TcODC, TcADC, and TcSAMDC may share signal transduction pathways and/or the stress induced signal induction pathways may converge at these three genes leading to similar although not identical patterns of expression. It is possible altering PA levels in cacao will result in enhanced tolerance to multiple stresses including drought and disease as has been demonstrated in other crops. Published by Elsevier Masson SAS.
Pathogenic diseases represent a major constraint to the growth and yield of cacao (Theobroma caca... more Pathogenic diseases represent a major constraint to the growth and yield of cacao (Theobroma cacao L.). Ongoing research on model plant systems has revealed that defense responses are activated via signaling pathways mediated by endogenous signaling molecules such as salicylic acid, jasmonic acid and ethylene. Activation of plant defenses is associated with changes in the expression of large numbers of genes. To gain a better understanding of defense responses in cacao, we have employed suppressive subtractive hybridization (SSH) cDNA libraries, macroarray hybridization analysis, high throughput DNA sequencing and bioinformatics to identify cacao genes induced by these signaling molecules. Additionally, we investigated gene activation by a phytotoxic elicitor-like protein, Nep1. We have identified a unigene set of 1,256 members, including 330 members representing genes induced during the defense response.
Theobroma cacao (cacao) is cultivated in tropical climates and is exposed to drought stress. The ... more Theobroma cacao (cacao) is cultivated in tropical climates and is exposed to drought stress. The impact of the endophytic fungus Trichoderma hamatum isolate DIS 219b on cacao's response to drought was studied. Colonization by DIS 219b delayed drought-induced changes in stomatal conductance, net photosynthesis, and green fluorescence emissions. The altered expression of 19 expressed sequence tags (ESTs) (seven in leaves and 17 in roots with some overlap) by drought was detected using quantitative real-time reverse transcription PCR. Roots tended to respond earlier to drought than leaves, with the drought-induced changes in expression of seven ESTs being observed after 7 d of withholding water. Changes in gene expression in leaves were not observed until after 10 d of withholding water. DIS 219b colonization delayed the drought-altered expression of all seven ESTs responsive to drought in leaves by >3 d, but had less influence on the expression pattern of the drought-responsive ESTs in roots. DIS 219b colonization had minimal direct influence on the expression of drought-responsive ESTs in 32-d-old seedlings. By contrast, DIS 219b colonization of 9-d-old seedlings altered expression of drought-responsive ESTs, sometimes in patterns opposite of that observed in response to drought. Drought induced an increase in the concentration of many amino acids in cacao leaves, while DIS 219b colonization caused a decrease in aspartic acid and glutamic acid concentrations and an increase in alanine and g-aminobutyric acid concentrations. With or without exposure to drought conditions, colonization by DIS 219b promoted seedling growth, the most consistent effects being an increase in root fresh weight, root dry weight, and root water content. Colonized seedlings were slower to wilt in response to drought as measured by a decrease in the leaf angle drop. The primary direct effect of DIS 219b colonization was promotion of root growth, regardless of water status, and an increase in water content which it is proposed caused a delay in many aspects of the drought response of cacao.
A chimeric octopine synthase-neomycin phosphotransferase (ocs-nptII) gene was used to transform C... more A chimeric octopine synthase-neomycin phosphotransferase (ocs-nptII) gene was used to transform Chlamydomonas reinhardiii to kanamycin resistance. Southern hybridization using DNA isolated from one transformant, T6.1, indicated that the entire ocs-nptII gene and at least part of the plasmid were integrated into nuclear DNA. Neomycin phosphotransferase II activity has been detected in T6.1 cell extracts. Northern hybridizations, employing a radiolabeled ocs-nptII sequence, revealed a T6.1 transcript of approximately the same size as a homologous transcript isolated from E. coli carrying the nptII gene. Although T6.1 is an extremely rare example of a stable C. reinhardtii transformant, its occurrence nevertheless indicates that bacterial genes can be expressed in the nucleus of the alga.
Theobroma cacao (cacao) suffers severe yield losses in many major production areas due to fungus-... more Theobroma cacao (cacao) suffers severe yield losses in many major production areas due to fungus-induced diseases. Cacao supports a complex endophytic microbial community that offers candidates for biocontrol of cacao diseases. Endophytic isolates of Trichoderma species were isolated from the live sapwood of trunks of Theobroma species, pods of Theobroma species, and a liana (Banisteriopsis caapi). Fifteen isolates of Trichoderma, potentially representing seven species, were selected for characterization of the influence of seedling inoculation on the establishment of endophytic growth in cacao seedlings. An isolate of Colletotrichum gloeosporioides was also included. The isolates studied in vitro varied in their abilities to produce metabolites inhibitory to Moniliophthora roreri and in their abilities to parasitize M. roreri cultures. The five inoculation methods used were: (1) inoculation of germinating seed on agar plates; (2) plate inoculation followed by planting in sterile soil; (3) planting sterile seed in pre-inoculated soil; (4) inoculation of emerged seedlings at the soil surface; and (5) inoculation of emerged seedlings between the cotyledon and stem. All the isolates studied were able to colonize Theobroma cacao seedlings, but isolates DIS 110a (Trichoderma cf. harzianum), DIS 219b (T. hamatum), DIS 219f (T. harzianum), and TA (T. asperellum) were the most efficient across inoculation methods. These same isolates also caused moderate to severe discoloration of roots of cacao seedlings germinated on water agar plates. Isolates DIS 173a (T. spirale), DIS 185c (T. stromaticum), and Col (Colletotrichum gloeosporioides) were inefficient colonizers of cacao. Most of the isolates studied were able to establish an endophytic relationship with cacao by colonizing the above ground portions of the cacao seedling, and exploitation of this characteristic could lead to the development of novel biocontrol strategies for control of cacao diseases.
... the perceived effectiveness of a mycoherbicide undoubtedly differ depending on the cropping s... more ... the perceived effectiveness of a mycoherbicide undoubtedly differ depending on the cropping system being used. ... of EN-4 in all four soil types tested (Table 1). Although variable, populations of EN ... of EN-4 in the soil in both Ūeld experiments, and this treatment effect also varied ...
In order to deregulate arginine biosynthesis in Synechococcus sp. PCC7942, d-arginine-resistant c... more In order to deregulate arginine biosynthesis in Synechococcus sp. PCC7942, d-arginine-resistant cell lines were selected following ethyl methanesulfonate mutagenesis of wild-type (WT) cells. Three of these arginine-producing mutant (APM) cell lines, APM1, APM31 and APM40, were putative regulatory mutants based upon secretion of l-arginine into their growth medium. HPLC of lyophilized post-harvest supernatants of APM 31 and 40 resolved two
Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes fo... more Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized ste...
Moniliophthora roreri is the fungal pathogen that causes frosty pod rot (FPR) disease of Theobrom... more Moniliophthora roreri is the fungal pathogen that causes frosty pod rot (FPR) disease of Theobroma cacao L., the source of chocolate. FPR occurs in most of the cacao producing countries in the Western Hemisphere, causing yield losses up to 80%. Genetic diversity within the FPR pathogen population may allow the population to adapt to changing environmental conditions and adapt to enhanced resistance in the host plant. The present study developed single nucleotide polymorphism (SNP) markers from RNASeq results for 13 M. roreri isolates and validated the markers for their ability to reveal genetic diversity in an international M. roreri collection. The SNP resources reported herein represent the first study of RNA sequencing (RNASeq)-derived SNP validation in M. roreri and demonstrates the utility of RNASeq as an approach for de novo SNP identification in M. roreri. A total of 88 polymorphic SNPs were used to evaluate the genetic diversity of 172 M. roreri cacao isolates resulting in 37 distinct genotypes (including 14 synonymous groups). Absence of heterozygosity for the 88 SNP markers indicates reproduction in M. roreri is clonal and likely due to a homothallic life style. The upper Magdalena Valley of Colombia showed the highest levels of genetic diversity with 20 distinct genotypes of which 13 were limited to this region, and indicates this region as the possible center of origin for M. roreri.
Theobroma cacao pods were inoculated with meiospores of Moniliophthora roreri (Mr), a hemibiotrop... more Theobroma cacao pods were inoculated with meiospores of Moniliophthora roreri (Mr), a hemibiotrophic basidiomycete causing frosty pod rot. Pods were malformed 30 days after inoculation (DAI) and sporulation was observed 60 DAI. Glucose and asparagine concentrations decreased and mannitol and malonate increased in infected pods 30 DAI. By 60 DAI, most carbohydrates, amino acids, and organic acids were drastically reduced by infection. Mannitol and succinic acid levels increased 60 DAI and likely originated from Mr. RT-qPCR analysis of cacao ESTs indicated a strong response to infection 30 DAI in malformed pod. Evidence indicated that biotrophic hyphae colonized pods and a shift to necrotrophic growth occurred later (during the end stages of infection). Expression of cacao ESTs associated with plant hormone biosynthesis and action was altered. Changes in the expression of Mr ESTs in response to nutrient deficiency in pure culture were small. Changes in Mr gene expression patterns and levels of specific metabolites in necrotic sporulating pods 60 DAI compared to malformed pods 30 DAI indicated that the glyoxylate cycle of Mr was up regulated during the shift from biotrophic to necrotrophic phases of the disease cycle.
Drought can negatively impact pod production despite the fact that cacao production usually occur... more Drought can negatively impact pod production despite the fact that cacao production usually occurs in tropical areas having high rainfall. Polyamines (PAs) have been associated with the response of plants to drought in addition to their roles in responses to many other stresses. The constitutive and drought inducible expression patterns of genes encoding enzymes involved in PA biosynthesis were determined:
Endophytic isolates of Trichoderma species are being considered as biocontrol agents for diseases... more Endophytic isolates of Trichoderma species are being considered as biocontrol agents for diseases of Theobroma cacao (cacao). Gene expression was studied during the interaction between cacao seedlings and four endophytic Trichoderma isolates, T. ovalisporum-DIS 70a, T. hamatum-DIS 219b, T. harzianum-DIS 219f, and Trichoderma sp.-DIS 172ai. Isolates DIS 70a, DIS 219b, and DIS 219f were mycoparasitic on the pathogen Moniliophthora roreri, and DIS 172ai produced metabolites that inhibited growth of M. roreri in culture. ESTs (116) responsive to endophytic colonization of cacao were identified using differential display and their expression analyzed using macroarrays. Nineteen cacao ESTs and 17 Trichoderma ESTs were chosen for real-time quantitative PCR analysis. Seven cacao ESTs were induced during colonization by the Trichoderma isolates. These included putative genes for ornithine decarboxylase (P1), GST-like proteins (P4), zinc finger protein (P13), wound-induced protein (P26), EF-calcium-binding protein (P29), carbohydrate oxidase (P59), and an unknown protein (U4). Two plant ESTs, extensin-like protein (P12) and major intrinsic protein (P31), were repressed due to colonization. The plant gene expression profile was dependent on the Trichoderma isolate colonizing the cacao seedling. The fungal ESTs induced in colonized cacao seedlings also varied with the Trichoderma isolate used. The most highly induced fungal ESTs were putative glucosyl hydrolase family 2 (F3), glucosyl hydrolase family 7 (F7), serine protease (F11), and alcohol oxidase (F19). The pattern of altered gene expression suggests a complex system of genetic cross talk occurs between the cacao tree and Trichoderma isolates during the establishment of the endophytic association.
Yield of Theobroma cacao (cacao), the source of chocolate, is limited by disease and insect pests... more Yield of Theobroma cacao (cacao), the source of chocolate, is limited by disease and insect pests. Developmental stage influences the resistance of cacao leaves to disease and insect pests. Mechanical wounding, ethylene, and methyl jasmonate induce resistance to pests in many plant species. The effects of mechanical wounding, ethylene, and methyl jasmonate on gene expression was studied in cacao leaves at two developmental stages, young red (YR) and mature green (MG). Differential expression was observed for genes putatively encoding a DNA binding protein (TcWRKY-1), a protein regulating cell division (TcORFX-1), a Type III peroxidase (TcPer-1), an endo-1,4-b-glucanase (TcGlu-1), a class VII chitinase (TcChiB), a caffeine synthase (TcCaf-1), and a light-harvesting complex protein (TcLhca-1). Wounding induced TcWRKY-1 and TcORFX-1 in YR and MG leaves. Elevated TcPer-1 mRNA levels were detected in YR and MG leaves after wounding. Wounding induced TcChiB in YR leaves and repressed TcLhca-1 in MG leaves. Ethylene (12 mL L À1 ) induced TcPer-1 in YR and MG leaves but induced TcGlu-1 in MG leaves only. Ethylene repressed TcLhca-1 and TcCaf-1 in YR leaves. Ethylene repressed TcLhca-1 and TcChiB in MG leaves. Methyl jasmonate (0.2 mM) induced TcCaf-1 and TcChiB in YR leaves and TcPer-1 and TcChiB in MG leaves. Ethylene/methyl jasmonate combined induced TcChiB in YR leaves and TcGlu-1 in MG leaves. 1-Methylcyclopropene, an inhibitor of ethylene action, blocked ethylene induced responses but did not block responses to wounding or methyl jasmonate. The cacao response to wounding, ethylene, and/or methyl jasmonate was influenced by developmental stage. Cross-talk between ethylene and methyl jasmonate action on cacao gene expression resulted in synergistic and antagonistic responses. It is critical to account for tissue developmental stage when studying the molecular responses of cacao to mechanical wounding, ethylene, and methyl jasmonate. The constituative and inducible defense strategies used by cacao are dependent on the developmental stage of the tissues involved. #
Developmental expression of stress response genes in Theobroma cacao leaves and their response to... more Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 and a compatible infection by Phytophthora megakarya were studied. Ten genes were selected to represent genes involved in defense TcGlu1, TcChiB,, gene regulation (TcWRKY-1 and TcORFX-1), cell wall development (TcCou-1, TcPer-1, and TcGlu-1), or energy production (TcLhca-1 and TcrbcS). Leaf development was separated into unexpanded (UE), young red (YR), immature green (IG), and mature green (MG). Our data indicates that the constitutive defense mechanisms used by cacao leaves differ between different developmental stages. TcWRKY-1 and TcChiB were highly expressed in MG leaves, and TcPer-1, TcGlu-1, and TcCou-1 were highly expressed inYR leaves. TcGlu1,3 was highly expressed in UE and YR leaves, TcCaf-1 was highly expressed in UE leaves, and TcLhca-1 and TcrbcS were highly expressed in IG and MG leaves. NEP1 encodes the necrosis inducing protein Nep1 produced by Fusarium oxysporum and has orthologs in Phytophthora species. Nep1 caused cellular necrosis on MG leaves and young pods within 24 h of application. Necrosis was observed on YR leaves 10 days after treatment. Expression of TcWRKY-1, TcORFX-1, TcPer-1, and TcGlu-1 was enhanced and TcLhca-1 and TcrbcS were repressed in MG leaves after Nep1 treatment. Expression of TcWRKY-1 and TcORFX-1 was enhanced in YR leaves after Nep1 treatment. Infection of MG leaf disks by P. megakarya zoospores enhanced expression of TcGlu-1, TcWRKY-1, and TcPer-1 and repressed expression of TcChiB, TcLhca-1 and TcrbcS. Five of the six genes that were responsive to Nep1 were responsive to infection by P. megakarya. Susceptibility of T. cacao to P. megakarya includes altered plant gene expression and phytotoxic molecules like Nep1 may contribute to susceptibility. Published by Elsevier SAS.
Drought can negatively impact pod production despite the fact that cacao production usually occur... more Drought can negatively impact pod production despite the fact that cacao production usually occurs in tropical areas having high rainfall. Polyamines (PAs) have been associated with the response of plants to drought in addition to their roles in responses to many other stresses. The constitutive and drought inducible expression patterns of genes encoding enzymes involved in PA biosynthesis were determined: an ornithine decarboxylase (TcODC ), an arginine decarboxylase (TcADC ), an S-adenosylmethionine decarboxylase (TcSAMDC ), a spermidine synthase (TcSPDS ), and a spermine synthase (TcSPMS ). Expression analysis using quantitative real-time reverse transcription-PCR (QPCR) results showed that the PA biosynthesis genes were expressed in all plant tissues examined. Constitutive expression of PA biosynthesis genes was generally highest in mature leaves and open flowers. Expression of TcODC, TcADC, and TcSAMDC was induced with the onset of drought and correlated with changes in stomatal conductance, photosynthesis, photosystem II efficiency, leaf water potential and altered emission of blue-green fluorescence from cacao leaves. Induction of TcSAMDC in leaves was most closely correlated with changes in water potential. The earliest measured responses to drought were enhanced expression of TcADC and TcSAMDC in roots along with decreases in stomatal conductance, photosynthesis, and photosystem II efficiency. Elevated levels of putrescine, spermidine, and spermine were detected in cacao leaves 13 days after the onset of drought. Expression of all five PA associated transcripts was enhanced (1.5e3-fold) in response to treatment with abscisic acid. TcODC and TcADC, were also responsive to mechanical wounding, infection by Phytophthora megakarya (a causal agent of black pod disease in cacao), the necrosis-and ethylene-inducing protein (Nep1) of Fusarium oxysporum, and flower abscission. TcSAMDC expression was responsive to all stresses except flower abscission. TcODC, although constitutively expressed at much lower levels than TcADC, TcSAMDC, TcSPDS, and TcSPMS, was highly inducible by the fungal protein Nep1 (135-fold) and the cacao pathogen Phytophthora megakarya (671-fold). The full length cDNA for ODC was cloned and characterized. Among the genes studied, TcODC, TcADC, and TcSAMDC were most sensitive to induction by drought in addition to other abiotic and biotic stresses. TcODC, TcADC, and TcSAMDC may share signal transduction pathways and/or the stress induced signal induction pathways may converge at these three genes leading to similar although not identical patterns of expression. It is possible altering PA levels in cacao will result in enhanced tolerance to multiple stresses including drought and disease as has been demonstrated in other crops. Published by Elsevier Masson SAS.
Pathogenic diseases represent a major constraint to the growth and yield of cacao (Theobroma caca... more Pathogenic diseases represent a major constraint to the growth and yield of cacao (Theobroma cacao L.). Ongoing research on model plant systems has revealed that defense responses are activated via signaling pathways mediated by endogenous signaling molecules such as salicylic acid, jasmonic acid and ethylene. Activation of plant defenses is associated with changes in the expression of large numbers of genes. To gain a better understanding of defense responses in cacao, we have employed suppressive subtractive hybridization (SSH) cDNA libraries, macroarray hybridization analysis, high throughput DNA sequencing and bioinformatics to identify cacao genes induced by these signaling molecules. Additionally, we investigated gene activation by a phytotoxic elicitor-like protein, Nep1. We have identified a unigene set of 1,256 members, including 330 members representing genes induced during the defense response.
Theobroma cacao (cacao) is cultivated in tropical climates and is exposed to drought stress. The ... more Theobroma cacao (cacao) is cultivated in tropical climates and is exposed to drought stress. The impact of the endophytic fungus Trichoderma hamatum isolate DIS 219b on cacao's response to drought was studied. Colonization by DIS 219b delayed drought-induced changes in stomatal conductance, net photosynthesis, and green fluorescence emissions. The altered expression of 19 expressed sequence tags (ESTs) (seven in leaves and 17 in roots with some overlap) by drought was detected using quantitative real-time reverse transcription PCR. Roots tended to respond earlier to drought than leaves, with the drought-induced changes in expression of seven ESTs being observed after 7 d of withholding water. Changes in gene expression in leaves were not observed until after 10 d of withholding water. DIS 219b colonization delayed the drought-altered expression of all seven ESTs responsive to drought in leaves by >3 d, but had less influence on the expression pattern of the drought-responsive ESTs in roots. DIS 219b colonization had minimal direct influence on the expression of drought-responsive ESTs in 32-d-old seedlings. By contrast, DIS 219b colonization of 9-d-old seedlings altered expression of drought-responsive ESTs, sometimes in patterns opposite of that observed in response to drought. Drought induced an increase in the concentration of many amino acids in cacao leaves, while DIS 219b colonization caused a decrease in aspartic acid and glutamic acid concentrations and an increase in alanine and g-aminobutyric acid concentrations. With or without exposure to drought conditions, colonization by DIS 219b promoted seedling growth, the most consistent effects being an increase in root fresh weight, root dry weight, and root water content. Colonized seedlings were slower to wilt in response to drought as measured by a decrease in the leaf angle drop. The primary direct effect of DIS 219b colonization was promotion of root growth, regardless of water status, and an increase in water content which it is proposed caused a delay in many aspects of the drought response of cacao.
A chimeric octopine synthase-neomycin phosphotransferase (ocs-nptII) gene was used to transform C... more A chimeric octopine synthase-neomycin phosphotransferase (ocs-nptII) gene was used to transform Chlamydomonas reinhardiii to kanamycin resistance. Southern hybridization using DNA isolated from one transformant, T6.1, indicated that the entire ocs-nptII gene and at least part of the plasmid were integrated into nuclear DNA. Neomycin phosphotransferase II activity has been detected in T6.1 cell extracts. Northern hybridizations, employing a radiolabeled ocs-nptII sequence, revealed a T6.1 transcript of approximately the same size as a homologous transcript isolated from E. coli carrying the nptII gene. Although T6.1 is an extremely rare example of a stable C. reinhardtii transformant, its occurrence nevertheless indicates that bacterial genes can be expressed in the nucleus of the alga.
Theobroma cacao (cacao) suffers severe yield losses in many major production areas due to fungus-... more Theobroma cacao (cacao) suffers severe yield losses in many major production areas due to fungus-induced diseases. Cacao supports a complex endophytic microbial community that offers candidates for biocontrol of cacao diseases. Endophytic isolates of Trichoderma species were isolated from the live sapwood of trunks of Theobroma species, pods of Theobroma species, and a liana (Banisteriopsis caapi). Fifteen isolates of Trichoderma, potentially representing seven species, were selected for characterization of the influence of seedling inoculation on the establishment of endophytic growth in cacao seedlings. An isolate of Colletotrichum gloeosporioides was also included. The isolates studied in vitro varied in their abilities to produce metabolites inhibitory to Moniliophthora roreri and in their abilities to parasitize M. roreri cultures. The five inoculation methods used were: (1) inoculation of germinating seed on agar plates; (2) plate inoculation followed by planting in sterile soil; (3) planting sterile seed in pre-inoculated soil; (4) inoculation of emerged seedlings at the soil surface; and (5) inoculation of emerged seedlings between the cotyledon and stem. All the isolates studied were able to colonize Theobroma cacao seedlings, but isolates DIS 110a (Trichoderma cf. harzianum), DIS 219b (T. hamatum), DIS 219f (T. harzianum), and TA (T. asperellum) were the most efficient across inoculation methods. These same isolates also caused moderate to severe discoloration of roots of cacao seedlings germinated on water agar plates. Isolates DIS 173a (T. spirale), DIS 185c (T. stromaticum), and Col (Colletotrichum gloeosporioides) were inefficient colonizers of cacao. Most of the isolates studied were able to establish an endophytic relationship with cacao by colonizing the above ground portions of the cacao seedling, and exploitation of this characteristic could lead to the development of novel biocontrol strategies for control of cacao diseases.
... the perceived effectiveness of a mycoherbicide undoubtedly differ depending on the cropping s... more ... the perceived effectiveness of a mycoherbicide undoubtedly differ depending on the cropping system being used. ... of EN-4 in all four soil types tested (Table 1). Although variable, populations of EN ... of EN-4 in the soil in both Ūeld experiments, and this treatment effect also varied ...
In order to deregulate arginine biosynthesis in Synechococcus sp. PCC7942, d-arginine-resistant c... more In order to deregulate arginine biosynthesis in Synechococcus sp. PCC7942, d-arginine-resistant cell lines were selected following ethyl methanesulfonate mutagenesis of wild-type (WT) cells. Three of these arginine-producing mutant (APM) cell lines, APM1, APM31 and APM40, were putative regulatory mutants based upon secretion of l-arginine into their growth medium. HPLC of lyophilized post-harvest supernatants of APM 31 and 40 resolved two
Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes fo... more Trichoderma species are usually considered soil organisms that colonize plant roots, sometimes forming a symbiotic relationship. Recent studies demonstrate that Trichoderma species are also capable of colonizing the above ground tissues of Theobroma cacao (cacao) in what has been characterized as an endophytic relationship. Trichoderma species can be re-isolated from surface sterilized cacao stem tissue, including the bark and xylem, the apical meristem, and to a lesser degree from leaves. SEM analysis of cacao stems colonized by strains of four Trichoderma species (Trichoderma ovalisporum-DIS 70a, Trichoderma hamatum-DIS 219b, Trichoderma koningiopsis-DIS 172ai, or Trichoderma harzianum-DIS 219f) showed a preference for surface colonization of glandular trichomes versus non-glandular trichomes. The Trichoderma strains colonized the glandular trichome tips and formed swellings resembling appresoria. Hyphae were observed emerging from the glandular trichomes on surface sterilized ste...
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Papers by Mary Strem