Theobroma cacao (cacao) is a tropical understory tree with sensitivity to drought. Cacao responds... more Theobroma cacao (cacao) is a tropical understory tree with sensitivity to drought. Cacao responds to drought by decreases in net photosynthesis, PS II efficiency, stomatal conductance, water potential and changes in leaf florescence. Drought also alters cacao gene expression as well as leaf glucose and free amino acid content. In recent years an incredible diversity of fungal endophytes has been
Frosty pod rot (FPR) of Theobroma cacao (cacao) is caused by the hemibiotrophic fungus Moniliopht... more Frosty pod rot (FPR) of Theobroma cacao (cacao) is caused by the hemibiotrophic fungus Moniliophthora roreri. Cacao clones tolerant to FPR are being planted throughout Central America. To determine whether M. roreri shows a differential molecular response during successful infections of tolerant clones, we collected field-infected pods at all stages of symptomatology for two highly susceptible clones (Pound-7 and CATIE-1000) and three tolerant clones (UF-273, CATIE-R7 and CATIE-R4). Metabolite analysis was carried out on clones Pound-7, CATIE-1000, CATIE-R7 and CATIE-R4. As FPR progressed, the concentrations of sugars in pods dropped, whereas the levels of trehalose and mannitol increased. Associations between symptoms and fungal loads and some organic and amino acid concentrations varied depending on the clone. RNA-Seq analysis identified 873 M. roreri genes that were differentially expressed between clones, with the primary difference being whether the clone was susceptible or tolerant. Genes encoding transcription factors, heat shock proteins, transporters, enzymes modifying membranes or cell walls and metabolic enzymes, such as malate synthase and alternative oxidase, were differentially expressed. The differential expression between clones of 43 M. roreri genes was validated by real-time quantitative reverse transcription polymerase chain reaction. The expression profiles of some genes were similar in susceptible and tolerant clones (other than CATIE-R4) and varied with the biotrophic/necrotropic shift. Moniliophthora roreri genes associated with stress metabolism and responses to heat shock and anoxia were induced early in tolerant clones, their expression profiles resembling that of the necrotrophic phase. Moniliophthora roreri stress response genes, induced during the infection of tolerant clones, may benefit the fungus in overcoming cacao defense mechanisms.
Theobroma cacao (cacao) is a cash crop for farmers in many countries along the equator. Cocoa, pr... more Theobroma cacao (cacao) is a cash crop for farmers in many countries along the equator. Cocoa, produced from cacao beans, is the basis for the chocolate industry in the United States and the world. One of the most devastating cacao diseases, frosty pod rot, is caused by Moniliophthora roreri. M. roreri is found in most cacao production areas in South and Central America other than Brazil. Recently the taxonomic identity of M. roreri was clarified and a clear relationship between M. roreri and Moniliophthora perniciosa, causal agent of witches’ broom, was established. As part of this work, the genome of M. roreri was sequenced and its transcriptome determined. Molecular analysis of the M. roreri/cacao interaction indicates the fungus avoids a strong defense reaction from cacao during the biotrophic phase of disease and spreads throughout the pod tissue much like an endophyte. The cacao pod defense/stress responses intensify for at least 30 days after infection. Eventually the pod rot...
Young Theobroma cacao pods, known as cherelles, are commonly lost to physiological thinning known... more Young Theobroma cacao pods, known as cherelles, are commonly lost to physiological thinning known as cherelle wilt. Cherelles are susceptible to frosty pod rot caused by Moniliophthora roreri. We studied the cherelle wilt process and its impact on M. roreri infection using microscopic, metabolite, and gene expression analyses. Wilt was associated with increased levels of tricarboxylic acid cycle intermediaries and decreased levels of major metabolites. Expression changes of cacao ESTs in response to wilt suggest induction of the polyamine, ethylene, and jasmonic acid biosynthetic pathways and regulation of abscisic acid and cytokinin levels. M. roreriinfection caused little alteration of cherelle physiology. M. roreri responded to the late stage of wilt by altering the expression of M. roreri ESTs associated with metabolite detoxification and host tissue degradation. The environment of the wilting cherelles may truncate the disease cycle of frosty pod rot, by limiting M. roreri sporulation and stopping the lifecycle.
Responses to drought were studied using two maize inbred lines (B76 and B106) and a commercial ma... more Responses to drought were studied using two maize inbred lines (B76 and B106) and a commercial maize hybrid (Zea mays L. cv. Silver Queen) with differing resistance to abiotic stress. Maize seedlings were grown in pots in controlled environment chambers for 17 days and watering was withheld from one half the plants for an additional 11 days. On the final treatment date, leaf water potentials did not differ among genotypes and were –0.84 and –1.49 MPa in the water sufficient and insufficient treatments, respectively. Greater rates of CO 2 assimilation were retained by the stress tolerant maize inbred line, B76, in comparison to the other two genotypes 11 days after watering was withheld. Rates of CO 2 assimilation for all three genotypes were unaffected by decreasing the measurement O 2 concentration from 21 to 2% (v/v). Activities of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), and NADP malate dehydrogenase were inhibited from 25 to 49% by the water deficienc...
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:
In recent years, carbon dioxide concentration [CO 2 ] in the atmosphere has risen to 370 µmol mol... more In recent years, carbon dioxide concentration [CO 2 ] in the atmosphere has risen to 370 µmol mol -l , with levels expected to double by the end of the 21 st century. A climatically-controlled greenhouse experiment was undertaken to assess the influence of [CO 2 ] and photosynthetic photon flux density (PPFD) on the growth, mineral nutrient uptake and mineral nutrient use efficiency parameters of cacao (Theobroma cacao L) in its early growth stages. Plants were grown in two greenhouses and maintained at two levels of [CO 2 ] (380 and 700 µmol mol -l). In each greenhouse, three levels of PPFD (65, 190, 1050 µmol m -2 s -1) were achieved by constructing mini shade frames covered with various layers of plastic shade cloth. Plants were grown for 57 days. At all levels of PPFD, with few exceptions, increasing [CO 2 ] tended to increase shoot and root growth parameters (dry wt. of roots, stem and leaves, stem height, leaf area, shoot/root ratio, leaf area ratio and relative growth rate). ...
Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom) are hemibio... more Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom) are hemibiotrophs causing disease in Theobroma cacao (cacao). Both pathogens have a prolonged biotrophic phase (typically greater than 45 days). During the biotrophic phase there is no evidence the pathogens form specialized cell wall penetration structures, instead surviving between cells. The mycelial morphology changes with the onset of necrosis along with a change in nuclear state from haploid to dikaryotic. M. roreri causes disease on pods and does not form basidiocarps while M. perniciosa forms basidiocarps and infects pods, flower cushions, and stems. We sequenced the M. roreri genome and transcriptome. The initial susceptible interaction between M. roreri and cacao is characterized by limited changes in cacao gene expression and slow progressive pod colonization. Late in the biotrophic phase (30 days after infection), a significant cacao stress response occurs along with limited changes in ...
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...
The ethylene biosynthesis-inducing xylanase (EIX) is known to be a potent elicitor of ethylene bi... more The ethylene biosynthesis-inducing xylanase (EIX) is known to be a potent elicitor of ethylene biosynthesis and other responses when applied to leaf tissue of Nicotiana tabacum L. cv Xanthi. In contrast, leaf tissue of the tobacco cultivar Hicks was insensitive to EIX at concentrations 100-fold higher than was needed to elicit responses from Xanthi. Cell-suspension cultures of Xanthi and Hicks showed similar differences in sensitivity to EIX. Equivalent levels of ethylene production were elicited in leaf discs of both cultivars after treatment with CuSO4. The F1 and Xanthi backcross progeny of Hicks and Xanthi crosses were all sensitive to EIX, whereas the F2 and Hicks backcross progeny segregated for sensitivity to EIX. Individual plants from the F2 and Hicks backcross that were insensitive to EIX produced only insensitive progeny when they were self-pollinated. Progeny from sensitive plants either segregated for sensitivity to EIX or produced all sensitive progeny (an F2 plant). S...
ABSTRACT The necrosis inducing extracellular protein Nep1 is produced by Fusarium oxysporum f. sp... more ABSTRACT The necrosis inducing extracellular protein Nep1 is produced by Fusarium oxysporum f. sp. erythroxyli in liquid culture. NEP1, the Nep1 protein structural gene, was disrupted in F. oxysporum f. sp. erythroxyli isolate EN-4 by gene replacement using polyethylene glycol (PEG)-mediated transformation. NEP1 disruption was verified by polymerase chain reaction (PCR), Southern blot, and northern blot analysis. NEP1-disrupted transformants failed to produce Nep1 in liquid culture. NEP1 disruption did not affect the pathogenicity of isolate EN-4 toward Erythroxylum coca. Transformation of isolate EN-4 with construct pPB-FO11-45 carrying NEP1 between the trpC promoter and terminator resulted in increased production of Nep1 in potato dextrose broth plus 1% casamino acids or Czapek-Dox broth plus 1% casamino acids but not in potato dextrose broth alone. Transformation of EN-4 with construct pPB-FO11-45 was verified by PCR and Southern blot analysis. Overexpression of NEP1 was confirmed by northern blot and Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. NEP1-overexpressing transformant 15 produced 64 to 128 times as much Nep1 as EN-4 wild type when grown in shake cultures. Transformants overexpressing Nep1 in liquid culture were no more or less pathogenic toward E. coca than wild-type isolates. Nep1 was not detected in E. coca seedlings infected with NEP1-overexpressing transformants or with EN-4 wild type. In large-scale fermentations of NEP1-overexpressing transformant 15, the amount of secreted protein including Nep1 was 15.1 times that of the wild-type EN-4, providing a ready source of Nep1 for future study.
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.
An increase in oxygen uptake has been previously described in plant cell suspensions treated with... more An increase in oxygen uptake has been previously described in plant cell suspensions treated with bacteria or bacterial elicitors. These studies, regarding oxygen uptake, have all been undertaken from the perspective of the host plant cell reacting to the invading pathogen. In contrast, here we describe and characterize an increase in oxygen uptake by bacterial cells in response to plant suspensions or autoclaved plant cell ®ltrates. Autoclaved plant cell ®ltrates stimulated bacterial oxygen uptake by as much as sevenfold within a few minutes after addition. This oxygen uptake response was proportional to both the concentration of the plant cell ®ltrate and the concentration of the bacteria. KCN inhibited the bacterial response, suggesting that bacterial respiration may be involved. Unlike the plant oxygen uptake response to bacteria, there was no concurrent H 2 O 2 accumulation and the NADPH oxidase inhibitor, DPI, had no eect on the bacterial response. Streptomycin, an inhibitor of bacterial protein synthesis, inhibited the bacterial oxygen uptake response to the plant cell ®ltrate. K-252, a protein kinase inhibitor that strongly inhibits the plant oxygen uptake response to bacteria, had no eect upon the bacterial oxygen uptake response. When potato/bacterial cell suspensions were pretreated with either streptomycin or K-252, the combined plant/bacterial oxygen uptake response was inhibited by 15 or 70 %, respectively. This indicates that as much as 15±30 % of the increased oxygen consumption during plant suspension cell/bacteria interactions may be attributable to bacteria, which comprise less than 1 % of the total cell mass.
A small assortment of microbial proteins have the ability to activate defense responses and induc... more A small assortment of microbial proteins have the ability to activate defense responses and induce necrosis in plant cells through cell signaling pathways. These proteins are of interest because of their potential use as bioherbicides and inducers of plant resistance in agriculture. A 24-kDa protein (Nep1) was purified from culture filtrates of Fusarium oxysporum, and the effects of this protein on weed leaves were investigated. This protein induced necrosis in detached leaves of Papaver somniferum, Lycopersicon esculentum, Malva neglecta, and Acroptilon repens when taken up through the petiole. The pattern and level of necrosis were dependent on the plant species. Treatment with Nep1 induced the production of ethylene in isolated leaves of various species, and the level of ethylene response was shown to be correlated to the concentration of the protein. Pretreating leaves of P. somniferum, L. esculentum, M. neglecta, and Cardaria draba with 100 l L -1 ethylene enhanced the protein induction of ethylene biosynthesis in those leaves. Application of Nep1 (200 nM) as a spray to intact plants of Abutilon theophrasti, P. somniferum, Centaurea solstitialis, Centaurea maculosa, and Sonchus oleraceus resulted in extensive necrosis of leaves within 48 h. The results of this research are supplemental to our understanding of the role of specific polypeptides in plant/microbe interactions and demonstrates for the first time that a fungal protein can cause extensive necrosis when applied to weed species as a foliar spray.
The fungal protein Nep1, produced by Fusarium oxysporum f. sp. erythroxyli in liquid culture, cau... more The fungal protein Nep1, produced by Fusarium oxysporum f. sp. erythroxyli in liquid culture, caused extensive necrosis to Centaurea maculosa when water solutions of Nep1 (5 g ml Ϫ1 ) and an organosilicone surfactant (1,1,1,3,5,5,5-heptamethyltrisiloxanyl propyl-methoxy-poly[ethylene oxide]) were applied as foliar sprays. Nep1 did not cause necrosis when applied with a nonionic surfactant or organosilicone surfactant plus unrefined corn oil. Plant age, protein concentration, organosilicone surfactant concentration, and the presence of a dew period influenced the amount of necrosis caused by Nep1. The addition of an 18-h dew period after treatment resulted in an increase of 10% or more in foliar necrosis at the 0.313 and 1.25 g ml Ϫ1 (0.40 and 1.62 g ai ha Ϫ1 ) Nep1 concentrations. Increasing the spray volume from 129 ml m Ϫ2 (1,291.3 L ha Ϫ1 ) to 516 ml m Ϫ2 (5,165.2 L ha Ϫ1 ) more than doubled the amount of foliar necrosis caused by the 0.313 g ml Ϫ1 (0.40 g ai ha Ϫ1 vs. 1.62 g ai ha Ϫ1 ) Nep1 concentration. A maximum necrosis rating of 95% was reached by 1.25 g ml Ϫ1 Nep1 applied at 516 ml m Ϫ2 (6.46 g ai ha Ϫ1 ) followed by an 18-h dew period. Nep1 (6.46 g ai ha Ϫ1 ) remained active when coapplied to Centaurea maculosa with the herbicides 2,4-D or glyphosate (0.13 to 2.58 kg ai ha Ϫ1 ), causing foliar necrosis prior to the herbicides killing Centaurea maculosa. An increase in the organosilicone surfactant concentration from 1 to 2 ml ai L Ϫ1 was required to achieve levels of Nep1-induced necrosis on Centaurea maculosa acclimated to direct sun comparable to levels achieved on greenhouse-grown plants. Repeated application of Nep1 (6.48 g ai ha Ϫ1 ) 3 wk after an initial treatment (6.48 g ai ha Ϫ1 ) prevented the recovery of acclimated Centaurea maculosa. Greater damage was caused to acclimated Centaurea maculosa when Nep1 was applied near the middle of the day (80% necrosis at 10:00 A.M. and 85% necrosis at 2:00 P.M.) compared to early or late in the day (25% necrosis at 6:00 A.M. and 10% necrosis at 6:00 P.M.).
Theobroma cacao (cacao) is a tropical understory tree with sensitivity to drought. Cacao responds... more Theobroma cacao (cacao) is a tropical understory tree with sensitivity to drought. Cacao responds to drought by decreases in net photosynthesis, PS II efficiency, stomatal conductance, water potential and changes in leaf florescence. Drought also alters cacao gene expression as well as leaf glucose and free amino acid content. In recent years an incredible diversity of fungal endophytes has been
Frosty pod rot (FPR) of Theobroma cacao (cacao) is caused by the hemibiotrophic fungus Moniliopht... more Frosty pod rot (FPR) of Theobroma cacao (cacao) is caused by the hemibiotrophic fungus Moniliophthora roreri. Cacao clones tolerant to FPR are being planted throughout Central America. To determine whether M. roreri shows a differential molecular response during successful infections of tolerant clones, we collected field-infected pods at all stages of symptomatology for two highly susceptible clones (Pound-7 and CATIE-1000) and three tolerant clones (UF-273, CATIE-R7 and CATIE-R4). Metabolite analysis was carried out on clones Pound-7, CATIE-1000, CATIE-R7 and CATIE-R4. As FPR progressed, the concentrations of sugars in pods dropped, whereas the levels of trehalose and mannitol increased. Associations between symptoms and fungal loads and some organic and amino acid concentrations varied depending on the clone. RNA-Seq analysis identified 873 M. roreri genes that were differentially expressed between clones, with the primary difference being whether the clone was susceptible or tolerant. Genes encoding transcription factors, heat shock proteins, transporters, enzymes modifying membranes or cell walls and metabolic enzymes, such as malate synthase and alternative oxidase, were differentially expressed. The differential expression between clones of 43 M. roreri genes was validated by real-time quantitative reverse transcription polymerase chain reaction. The expression profiles of some genes were similar in susceptible and tolerant clones (other than CATIE-R4) and varied with the biotrophic/necrotropic shift. Moniliophthora roreri genes associated with stress metabolism and responses to heat shock and anoxia were induced early in tolerant clones, their expression profiles resembling that of the necrotrophic phase. Moniliophthora roreri stress response genes, induced during the infection of tolerant clones, may benefit the fungus in overcoming cacao defense mechanisms.
Theobroma cacao (cacao) is a cash crop for farmers in many countries along the equator. Cocoa, pr... more Theobroma cacao (cacao) is a cash crop for farmers in many countries along the equator. Cocoa, produced from cacao beans, is the basis for the chocolate industry in the United States and the world. One of the most devastating cacao diseases, frosty pod rot, is caused by Moniliophthora roreri. M. roreri is found in most cacao production areas in South and Central America other than Brazil. Recently the taxonomic identity of M. roreri was clarified and a clear relationship between M. roreri and Moniliophthora perniciosa, causal agent of witches’ broom, was established. As part of this work, the genome of M. roreri was sequenced and its transcriptome determined. Molecular analysis of the M. roreri/cacao interaction indicates the fungus avoids a strong defense reaction from cacao during the biotrophic phase of disease and spreads throughout the pod tissue much like an endophyte. The cacao pod defense/stress responses intensify for at least 30 days after infection. Eventually the pod rot...
Young Theobroma cacao pods, known as cherelles, are commonly lost to physiological thinning known... more Young Theobroma cacao pods, known as cherelles, are commonly lost to physiological thinning known as cherelle wilt. Cherelles are susceptible to frosty pod rot caused by Moniliophthora roreri. We studied the cherelle wilt process and its impact on M. roreri infection using microscopic, metabolite, and gene expression analyses. Wilt was associated with increased levels of tricarboxylic acid cycle intermediaries and decreased levels of major metabolites. Expression changes of cacao ESTs in response to wilt suggest induction of the polyamine, ethylene, and jasmonic acid biosynthetic pathways and regulation of abscisic acid and cytokinin levels. M. roreriinfection caused little alteration of cherelle physiology. M. roreri responded to the late stage of wilt by altering the expression of M. roreri ESTs associated with metabolite detoxification and host tissue degradation. The environment of the wilting cherelles may truncate the disease cycle of frosty pod rot, by limiting M. roreri sporulation and stopping the lifecycle.
Responses to drought were studied using two maize inbred lines (B76 and B106) and a commercial ma... more Responses to drought were studied using two maize inbred lines (B76 and B106) and a commercial maize hybrid (Zea mays L. cv. Silver Queen) with differing resistance to abiotic stress. Maize seedlings were grown in pots in controlled environment chambers for 17 days and watering was withheld from one half the plants for an additional 11 days. On the final treatment date, leaf water potentials did not differ among genotypes and were –0.84 and –1.49 MPa in the water sufficient and insufficient treatments, respectively. Greater rates of CO 2 assimilation were retained by the stress tolerant maize inbred line, B76, in comparison to the other two genotypes 11 days after watering was withheld. Rates of CO 2 assimilation for all three genotypes were unaffected by decreasing the measurement O 2 concentration from 21 to 2% (v/v). Activities of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), and NADP malate dehydrogenase were inhibited from 25 to 49% by the water deficienc...
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:
In recent years, carbon dioxide concentration [CO 2 ] in the atmosphere has risen to 370 µmol mol... more In recent years, carbon dioxide concentration [CO 2 ] in the atmosphere has risen to 370 µmol mol -l , with levels expected to double by the end of the 21 st century. A climatically-controlled greenhouse experiment was undertaken to assess the influence of [CO 2 ] and photosynthetic photon flux density (PPFD) on the growth, mineral nutrient uptake and mineral nutrient use efficiency parameters of cacao (Theobroma cacao L) in its early growth stages. Plants were grown in two greenhouses and maintained at two levels of [CO 2 ] (380 and 700 µmol mol -l). In each greenhouse, three levels of PPFD (65, 190, 1050 µmol m -2 s -1) were achieved by constructing mini shade frames covered with various layers of plastic shade cloth. Plants were grown for 57 days. At all levels of PPFD, with few exceptions, increasing [CO 2 ] tended to increase shoot and root growth parameters (dry wt. of roots, stem and leaves, stem height, leaf area, shoot/root ratio, leaf area ratio and relative growth rate). ...
Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom) are hemibio... more Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches’ broom) are hemibiotrophs causing disease in Theobroma cacao (cacao). Both pathogens have a prolonged biotrophic phase (typically greater than 45 days). During the biotrophic phase there is no evidence the pathogens form specialized cell wall penetration structures, instead surviving between cells. The mycelial morphology changes with the onset of necrosis along with a change in nuclear state from haploid to dikaryotic. M. roreri causes disease on pods and does not form basidiocarps while M. perniciosa forms basidiocarps and infects pods, flower cushions, and stems. We sequenced the M. roreri genome and transcriptome. The initial susceptible interaction between M. roreri and cacao is characterized by limited changes in cacao gene expression and slow progressive pod colonization. Late in the biotrophic phase (30 days after infection), a significant cacao stress response occurs along with limited changes in ...
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...
The ethylene biosynthesis-inducing xylanase (EIX) is known to be a potent elicitor of ethylene bi... more The ethylene biosynthesis-inducing xylanase (EIX) is known to be a potent elicitor of ethylene biosynthesis and other responses when applied to leaf tissue of Nicotiana tabacum L. cv Xanthi. In contrast, leaf tissue of the tobacco cultivar Hicks was insensitive to EIX at concentrations 100-fold higher than was needed to elicit responses from Xanthi. Cell-suspension cultures of Xanthi and Hicks showed similar differences in sensitivity to EIX. Equivalent levels of ethylene production were elicited in leaf discs of both cultivars after treatment with CuSO4. The F1 and Xanthi backcross progeny of Hicks and Xanthi crosses were all sensitive to EIX, whereas the F2 and Hicks backcross progeny segregated for sensitivity to EIX. Individual plants from the F2 and Hicks backcross that were insensitive to EIX produced only insensitive progeny when they were self-pollinated. Progeny from sensitive plants either segregated for sensitivity to EIX or produced all sensitive progeny (an F2 plant). S...
ABSTRACT The necrosis inducing extracellular protein Nep1 is produced by Fusarium oxysporum f. sp... more ABSTRACT The necrosis inducing extracellular protein Nep1 is produced by Fusarium oxysporum f. sp. erythroxyli in liquid culture. NEP1, the Nep1 protein structural gene, was disrupted in F. oxysporum f. sp. erythroxyli isolate EN-4 by gene replacement using polyethylene glycol (PEG)-mediated transformation. NEP1 disruption was verified by polymerase chain reaction (PCR), Southern blot, and northern blot analysis. NEP1-disrupted transformants failed to produce Nep1 in liquid culture. NEP1 disruption did not affect the pathogenicity of isolate EN-4 toward Erythroxylum coca. Transformation of isolate EN-4 with construct pPB-FO11-45 carrying NEP1 between the trpC promoter and terminator resulted in increased production of Nep1 in potato dextrose broth plus 1% casamino acids or Czapek-Dox broth plus 1% casamino acids but not in potato dextrose broth alone. Transformation of EN-4 with construct pPB-FO11-45 was verified by PCR and Southern blot analysis. Overexpression of NEP1 was confirmed by northern blot and Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. NEP1-overexpressing transformant 15 produced 64 to 128 times as much Nep1 as EN-4 wild type when grown in shake cultures. Transformants overexpressing Nep1 in liquid culture were no more or less pathogenic toward E. coca than wild-type isolates. Nep1 was not detected in E. coca seedlings infected with NEP1-overexpressing transformants or with EN-4 wild type. In large-scale fermentations of NEP1-overexpressing transformant 15, the amount of secreted protein including Nep1 was 15.1 times that of the wild-type EN-4, providing a ready source of Nep1 for future study.
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.
An increase in oxygen uptake has been previously described in plant cell suspensions treated with... more An increase in oxygen uptake has been previously described in plant cell suspensions treated with bacteria or bacterial elicitors. These studies, regarding oxygen uptake, have all been undertaken from the perspective of the host plant cell reacting to the invading pathogen. In contrast, here we describe and characterize an increase in oxygen uptake by bacterial cells in response to plant suspensions or autoclaved plant cell ®ltrates. Autoclaved plant cell ®ltrates stimulated bacterial oxygen uptake by as much as sevenfold within a few minutes after addition. This oxygen uptake response was proportional to both the concentration of the plant cell ®ltrate and the concentration of the bacteria. KCN inhibited the bacterial response, suggesting that bacterial respiration may be involved. Unlike the plant oxygen uptake response to bacteria, there was no concurrent H 2 O 2 accumulation and the NADPH oxidase inhibitor, DPI, had no eect on the bacterial response. Streptomycin, an inhibitor of bacterial protein synthesis, inhibited the bacterial oxygen uptake response to the plant cell ®ltrate. K-252, a protein kinase inhibitor that strongly inhibits the plant oxygen uptake response to bacteria, had no eect upon the bacterial oxygen uptake response. When potato/bacterial cell suspensions were pretreated with either streptomycin or K-252, the combined plant/bacterial oxygen uptake response was inhibited by 15 or 70 %, respectively. This indicates that as much as 15±30 % of the increased oxygen consumption during plant suspension cell/bacteria interactions may be attributable to bacteria, which comprise less than 1 % of the total cell mass.
A small assortment of microbial proteins have the ability to activate defense responses and induc... more A small assortment of microbial proteins have the ability to activate defense responses and induce necrosis in plant cells through cell signaling pathways. These proteins are of interest because of their potential use as bioherbicides and inducers of plant resistance in agriculture. A 24-kDa protein (Nep1) was purified from culture filtrates of Fusarium oxysporum, and the effects of this protein on weed leaves were investigated. This protein induced necrosis in detached leaves of Papaver somniferum, Lycopersicon esculentum, Malva neglecta, and Acroptilon repens when taken up through the petiole. The pattern and level of necrosis were dependent on the plant species. Treatment with Nep1 induced the production of ethylene in isolated leaves of various species, and the level of ethylene response was shown to be correlated to the concentration of the protein. Pretreating leaves of P. somniferum, L. esculentum, M. neglecta, and Cardaria draba with 100 l L -1 ethylene enhanced the protein induction of ethylene biosynthesis in those leaves. Application of Nep1 (200 nM) as a spray to intact plants of Abutilon theophrasti, P. somniferum, Centaurea solstitialis, Centaurea maculosa, and Sonchus oleraceus resulted in extensive necrosis of leaves within 48 h. The results of this research are supplemental to our understanding of the role of specific polypeptides in plant/microbe interactions and demonstrates for the first time that a fungal protein can cause extensive necrosis when applied to weed species as a foliar spray.
The fungal protein Nep1, produced by Fusarium oxysporum f. sp. erythroxyli in liquid culture, cau... more The fungal protein Nep1, produced by Fusarium oxysporum f. sp. erythroxyli in liquid culture, caused extensive necrosis to Centaurea maculosa when water solutions of Nep1 (5 g ml Ϫ1 ) and an organosilicone surfactant (1,1,1,3,5,5,5-heptamethyltrisiloxanyl propyl-methoxy-poly[ethylene oxide]) were applied as foliar sprays. Nep1 did not cause necrosis when applied with a nonionic surfactant or organosilicone surfactant plus unrefined corn oil. Plant age, protein concentration, organosilicone surfactant concentration, and the presence of a dew period influenced the amount of necrosis caused by Nep1. The addition of an 18-h dew period after treatment resulted in an increase of 10% or more in foliar necrosis at the 0.313 and 1.25 g ml Ϫ1 (0.40 and 1.62 g ai ha Ϫ1 ) Nep1 concentrations. Increasing the spray volume from 129 ml m Ϫ2 (1,291.3 L ha Ϫ1 ) to 516 ml m Ϫ2 (5,165.2 L ha Ϫ1 ) more than doubled the amount of foliar necrosis caused by the 0.313 g ml Ϫ1 (0.40 g ai ha Ϫ1 vs. 1.62 g ai ha Ϫ1 ) Nep1 concentration. A maximum necrosis rating of 95% was reached by 1.25 g ml Ϫ1 Nep1 applied at 516 ml m Ϫ2 (6.46 g ai ha Ϫ1 ) followed by an 18-h dew period. Nep1 (6.46 g ai ha Ϫ1 ) remained active when coapplied to Centaurea maculosa with the herbicides 2,4-D or glyphosate (0.13 to 2.58 kg ai ha Ϫ1 ), causing foliar necrosis prior to the herbicides killing Centaurea maculosa. An increase in the organosilicone surfactant concentration from 1 to 2 ml ai L Ϫ1 was required to achieve levels of Nep1-induced necrosis on Centaurea maculosa acclimated to direct sun comparable to levels achieved on greenhouse-grown plants. Repeated application of Nep1 (6.48 g ai ha Ϫ1 ) 3 wk after an initial treatment (6.48 g ai ha Ϫ1 ) prevented the recovery of acclimated Centaurea maculosa. Greater damage was caused to acclimated Centaurea maculosa when Nep1 was applied near the middle of the day (80% necrosis at 10:00 A.M. and 85% necrosis at 2:00 P.M.) compared to early or late in the day (25% necrosis at 6:00 A.M. and 10% necrosis at 6:00 P.M.).
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