Metabolomic and Transcriptomic Profiling Provide Novel Insights into Fruit Ripening and Ripening Disorder Caused by 1-MCP Treatments in Papaya.

Treatment with 1-methylcyclopropylene (1-MCP) is an effective technique to preserve fruits, but inappropriate treatment with 1-MCP causes a ripening disorder (rubbery texture) in papaya fruit. In this study, a combined metabolomic and transcriptomic analysis was conducted to reveal the possible mechanism of the ripening disorder caused by unsuitable 1-MCP in papaya. A total of 203 differential accumulated metabolites (DAMs) were identified in the metabolome analysis. Only 24 DAMs were identified in the control (CK) vs. the 1-MCP 2 h group, and they were primarily flavonoids. Ninety and 89 DAMs were identified in the CK vs. 1-MCP 16 h and 1-MCP 2 h vs. 1-MCP 16 h groups, respectively, indicating that long-term 1-MCP treatment severely altered the metabolites during fruit ripening. 1-MCP 16 h treatment severely reduced the number of metabolites, which primarily consisted of flavonoids, lipids, phenolic acids, alkaloids, and organic acids. An integrated analysis of RNA-Seq and metabolomics showed that various energy metabolites for the tricarboxylic acid cycle were reduced by long-term treatment with 1-MCP, and the glycolic acid cycle was the most significantly affected, as well as the phenylpropane pathway. These results provide valuable information for fruit quality control and new insight into the ripening disorder caused by unsuitable treatment with 1-MCP in papaya.

Active coatings based on hydroxypropyl methylcellulose and silver nanoparticles to extend the papaya (Carica papaya L.) shelf life.

This study aimed to understand the effect of silver nanoparticles (AgNPs) on physiochemical properties of hydroxypropyl methylcellulose (HPMC) film-forming solutions (FFS) and nanocomposite films (NCF), as well as the efficacy of these materials to control the development of anthracnose caused by Colletotrichum gloeosporioides in papaya (Carica papaya L.). FFS were characterized by pH, particle size distribution, and rheology. In addition, thickness, morphology, water contact angle, barrier, chemical, crystallinity, thermal, and mechanical properties from NCF were investigated. The minimum inhibitory concentration of AgNPs against C. gloeosporioides was determined by in vitro test. FFS with 0.25 wt% of AgNPs were used as coatings in papayas inoculated with C. gloeosporioides. Finally, the physicochemical parameters were investigated during their storing up to 7 days at 10 °C, followed by 7 days at 20 °C. The presence of AgNPs impacted the thickness, morphology, moisture content, chemical bonds, crystalline structure, and thermal properties of films. Coatings with 0.25 wt% of AgNPs reduced the incidence and severity of C. gloeosporioides and avoided the weight loss of papayas during storing. The ripening of papaya occurred naturally, showing that the coating only delayed this process. Thus, HPMC-AgNPs coating can be an alternative to extend the papaya shelf life.

Roles of transcription factor SQUAMOSA promoter binding protein-like gene family in papaya (Carica papaya) development and ripening.

SQUAMOSA promoter binding protein-like (SPL) family plays vital regulatory roles in plant growth and development. The SPL family in climacteric fruit Carica papaya has not been reported. This study identified 14 papaya SPLs (CpSPL) from papaya genome and analyzed their sequence features, phylogeny, intron/exon structure, conserved motif, miR156-mediated posttranscriptional regulation, and expression patterns. 14 CpSPLs were clustered into 8 groups, and two distinct expression patterns were revealed for miR156-targeted and nontargeted CpSPLs in different tissues and fruit development stages. The expression changes of CpSPLs in ethephon and 1-MCP treated fruit during ripening suggested that the CpSPLs guided by CpmiR156 play crucial roles in ethylene signaling pathway. This study sheds light on the new function of SPL family in fruit development and ripening, providing insights on understanding evolutionary divergence of the members of SPL family among plant species.

Biological control of Erwinia mallotivora, the causal agent of papaya dieback disease by indigenous seed-borne endophytic lactic acid bacteria consortium.

Dieback disease caused by Erwinia mallotivora is a major threat to papaya plantation in Malaysia. The current study was conducted to evaluate the potential of endophytic lactic acid bacteria (LAB) isolated from papaya seeds for disease suppression of papaya dieback. Two hundred and thirty isolates were screened against E. mallotivora BT-MARDI, and the inhibitory activity of the isolates against the pathogen was ranging from 11.7-23.7 mm inhibition zones. The synergistic experiments revealed that combination of W. cibaria PPKSD19 and Lactococcus lactis subsp. lactis PPSSD39 increased antibacterial activity against the pathogen. The antibacterial activity was partially due to the production of bacteriocin-like inhibitory substances (BLIS). The nursery experiment confirmed that the application of bacterial consortium W. cibaria PPKSD19 and L. lactis subsp. lactis PPSSD39 significantly reduced disease severity to 19% and increased biocontrol efficacy to 69% of infected papaya plants after 18 days of treatment. This study showed that W. cibaria PPKSD19 and L. lactis subsp. lactis PPSSD39 are potential candidate as biocontrol agents against papaya dieback disease.

Application of coatings formed by chitosan and Mentha essential oils to control anthracnose caused by Colletotrichum gloesporioides and C. brevisporum in papaya (Carica papaya L.) fruit.

This study investigated the efficacy of coatings formed by chitosan (Chi) and Mentha piperita L. (MPEO) or M. × villosa Huds (MVEO) essential oil to control the development of antrachnnose in papaya fruit caused by Colletotrichum gloeosporioides and C. brevisporum. Chi (2.5-10 mg/mL), MPEO and MVEO (0.15-1.25 µL/mL) alone effectively inhibited the growth of C. gloeosporioides and C. brevisporum isolates in laboratory media. Combinations of Chi (5 and 7.5 mg/mL) and MPEO or MVEO (0.15-1.25 mL/mL) inhibited the growth of Colletotrichum isolates and mostly presented additive or synergistic interactions. Development of anthracnose lesions caused by C. gloeosporioides and C. brevisporum isolates was reduced by coatings formed by Chi (5 mg/mL) and MPEO or MVEO (0.3-1.25 µL/mL) combinations during storage (10 days, 25 ±â€¯0.5 °C). Decreases in anthracnose lesion development in papaya coated with Chi (5 mg/mL) and MPEO or MVEO (0.6 and 1.25 µL/mL) were similar or higher than those caused by a comercial fungicides formulation. The application of coatings formed by combinations of selected Chi and MPEO or MVEO concentrations could be considered an alternative strategy to control papaya anthracnose caused by C. gloeosporioides and C. brevisporum.

The effects of an osmoregulator, carbohydrates and polyol on maturation and germination of 'Golden THB' papaya somatic embryos.

This study evaluated the effect of osmoregulators and carbohydrates on the maturation and germination of somatic embryos of papaya 'Golden THB'. Cotyledon explants from papaya seedlings germinated in vitro on basal MS medium were cultured on somatic embryogenesis induction medium (IM) containing MS salts, myo-inositol, sucrose, agar and p-chlorophenoxyacetic acid. After 50 days, embryogenic calli were transferred onto maturation media (MM) for 45 additional days. For experiment 1, a MS-based medium supplemented with abscisic acid, activated charcoal and concentrations of PEG 6000 (0; 40; 50; 60 and 70 g L-1) was used, whereas for experiment 2 malt extract concentrations (0; 0.1; 0.2; 0.3 and 0.4 g L-1) were assessed. The normal cotyledonary somatic embryos produced in experiment 2 were transferred to the germination medium (GM). The GM consisted of full-strength MS medium, sucrose, agar and was supplemented with myo-inositol at varying concentrations (0; 0.275; 0.55 and 0.825 mM). The PEG concentrations tested impaired the maturation of 'Golden THB' papaya somatic embryos. The MM, supplemented with malt extract at 0.153 g L-1, promoted the greatest development of normal somatic embryos (18.28 SE calli-1), that is, two cotyledonary leaves produced 36.56 SE calli-1. The supplementation with 0.45 mM myo-inositol provided the highest germination percentage (47.42%) and conversion to emblings.

Transcriptome analysis provides insights into the delayed sticky disease symptoms in Carica papaya.

KEY MESSAGE: Global gene expression analysis indicates host stress responses, mainly those mediated by SA, associated to the tolerance to sticky disease symptoms at pre-flowering stage in Carica papaya. Carica papaya plants develop the papaya sticky disease (PSD) as a result of the combined infection of papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), or PMeV complex. PSD symptoms appear only after C. papaya flowers. To understand the mechanisms involved in this phenomenon, the global gene expression patterns of PMeV complex-infected C. papaya at pre-and post-flowering stages were assessed by RNA-Seq. The result was 633 and 88 differentially expressed genes at pre- and post-flowering stages, respectively. At pre-flowering stage, genes related to stress and transport were up-regulated while metabolism-related genes were down-regulated. It was observed that induction of several salicylic acid (SA)-activated genes, including PR1, PR2, PR5, WRKY transcription factors, ROS and callose genes, suggesting SA signaling involvement in the delayed symptoms. In fact, pre-flowering C. papaya treated with exogenous SA showed a tendency to decrease the PMeV and PMeV2 loads when compared to control plants. However, pre-flowering C. papaya also accumulated transcripts encoding a NPR1-inhibitor (NPR1-I/NIM1-I) candidate, genes coding for UDP-glucosyltransferases (UGTs) and several genes involved with ethylene pathway, known to be negative regulators of SA signaling. At post-flowering, when PSD symptoms appeared, the down-regulation of PR-1 encoding gene and the induction of BSMT1 and JA metabolism-related genes were observed. Hence, SA signaling likely operates at the pre-flowering stage of PMeV complex-infected C. papaya inhibiting the development of PSD symptoms, but the induction of its negative regulators prevents the full-scale and long-lasting tolerance.

Somatic embryogenesis in Carica papaya as affected by auxins and explants, and morphoanatomical-related aspects

ABSTRACT The aim of this study was to evaluate somatic embryogenesis in juvenile explants of the THB papaya cultivar. Apical shoots and cotyledonary leaves were inoculated in an induction medium composed of different concentrations of 2,4-D (6, 9, 12, 15 and 18 µM) or 4-CPA (19, 22, 25, 28 and 31 µM). The embryogenic calluses were transferred to a maturation medium for 30 days. Histological analysis were done during the induction and scanning electron microscopy after maturing. For both types of auxin, embryogenesis was achieved at higher frequencies with cotyledonary leaves incubated in induction medium than with apical shoots; except for callogenesis. The early-stage embryos (e.g., globular or heart-shape) predominated. Among the auxins, best results were observed in cotyledonary leaves induced with 4-CPA (25 µM). Histological analyses of the cotyledonary leaf-derived calluses confirmed that the somatic embryos (SEs) formed from parenchyma cells, predominantly differentiated via indirect and multicellular origin and infrequently via synchronized embryogenesis. The secondary embryogenesis was observed during induction and maturation phases in papaya THB cultivar. The combination of ABA (0.5 µM) and AC (15 g L-1) in maturation medium resulted in the highest somatic embryogenesis induction frequency (70 SEs callus-1) and the lowest percentage of early germination (4%).

Somatic embryogenesis in Carica papaya as affected by auxins and explants, and morphoanatomical-related aspects.

The aim of this study was to evaluate somatic embryogenesis in juvenile explants of the THB papaya cultivar. Apical shoots and cotyledonary leaves were inoculated in an induction medium composed of different concentrations of 2,4-D (6, 9, 12, 15 and 18 µM) or 4-CPA (19, 22, 25, 28 and 31 µM). The embryogenic calluses were transferred to a maturation medium for 30 days. Histological analysis were done during the induction and scanning electron microscopy after maturing. For both types of auxin, embryogenesis was achieved at higher frequencies with cotyledonary leaves incubated in induction medium than with apical shoots; except for callogenesis. The early-stage embryos (e.g., globular or heart-shape) predominated. Among the auxins, best results were observed in cotyledonary leaves induced with 4-CPA (25 µM). Histological analyses of the cotyledonary leaf-derived calluses confirmed that the somatic embryos (SEs) formed from parenchyma cells, predominantly differentiated via indirect and multicellular origin and infrequently via synchronized embryogenesis. The secondary embryogenesis was observed during induction and maturation phases in papaya THB cultivar. The combination of ABA (0.5 µM) and AC (15 g L-1) in maturation medium resulted in the highest somatic embryogenesis induction frequency (70 SEs callus-1) and the lowest percentage of early germination (4%).

A Phytophthora palmivora Extracellular Cystatin-Like Protease Inhibitor Targets Papain to Contribute to Virulence on Papaya.

Papaya fruits, stems, and leaves are rich in papain, a cysteine protease that has been shown to mediate plant defense against pathogens and insects. Yet the oomycete Phytophthora palmivora is a destructive pathogen that infects all parts of papaya plants, suggesting that it has evolved cysteine protease inhibitors to inhibit papain to enable successful infection. Out of five putative extracellular cystatin-like cysteine protease inhibitors (PpalEPICs) from P. palmivora transcriptomic sequence data, PpalEPIC8 appeared to be unique to P. palmivora and was highly induced during infection of papaya. Purified recombinant PpalEPIC8 strongly inhibited papain enzyme activity, suggesting that it is a functional cysteine protease inhibitor. Homozygous PpalEPIC8 mutants were generated using CRISPR/Cas9-mediated gene editing via Agrobacterium-mediated transformation (AMT). Increased papain sensitivity of in-vitro growth and reduced pathogenicity during infection of papaya fruits were observed for the mutants compared with the wild-type strain, suggesting that PpalEPIC8, indeed, plays a role in P. palmivora virulence by inhibiting papain. This study provided genetic evidence demonstrating that plant-pathogenic oomycetes secrete cystatins as important weapons to invade plants. It also established an effective gene-editing system for P. palmivora by the combined use of CRISPR/Cas9 and AMT, which is expected to be applicable to other oomycetes.

Isolation of ripening-related genes from ethylene/1-MCP treated papaya through RNA-seq.

BACKGROUND: Since papaya is a typical climacteric fruit, exogenous ethylene (ETH) applications can induce premature and quicker ripening, while 1-methylcyclopropene (1-MCP) slows down the ripening processes. Differential gene expression in ETH or 1-MCP-treated papaya fruits accounts for the ripening processes. To isolate the key ripening-related genes and better understand fruit ripening mechanisms, transcriptomes of ETH or 1-MCP-treated, and non-treated (Control Group, CG) papaya fruits were sequenced using Illumina Hiseq2500. RESULTS: A total of 18,648 (1-MCP), 19,093 (CG), and 15,321 (ETH) genes were detected, with the genes detected in the ETH-treatment being the least. This suggests that ETH may inhibit the expression of some genes. Based on the differential gene expression (DGE) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, 53 fruit ripening-related genes were selected: 20 cell wall-related genes, 18 chlorophyll and carotenoid metabolism-related genes, four proteinases and their inhibitors, six plant hormone signal transduction pathway genes, four transcription factors, and one senescence-associated gene. Reverse transcription quantitative PCR (RT-qPCR) analyses confirmed the results of RNA-seq and verified that the expression pattern of six genes is consistent with the fruit senescence process. Based on the expression profiling of genes in carbohydrate metabolic process, chlorophyll metabolism pathway, and carotenoid metabolism pathway, the mechanism of pulp softening and coloration of papaya was deduced and discussed. We illustrate that papaya fruit softening is a complex process with significant cell wall hydrolases, such as pectinases, cellulases, and hemicellulases involved in the process. Exogenous ethylene accelerates the coloration of papaya changing from green to yellow. This is likely due to the inhibition of chlorophyll biosynthesis and the α-branch of carotenoid metabolism. Chy-b may play an important role in the yellow color of papaya fruit. CONCLUSIONS: Comparing the differential gene expression in ETH/1-MCP-treated papaya using RNA-seq is a sound approach to isolate ripening-related genes. The results of this study can improve our understanding of papaya fruit ripening molecular mechanism and reveal candidate fruit ripening-related genes for further research.

Ficus carica L. (Moraceae): an ancient source of food and health.

Since early in the man history, common fig was appreciated as food and for its medicinal properties. This review explores some aspects about the importance of Ficus carica L., an amazing and ancient source of medicines and food. Topics regarding chemistry, biological activity, ethno-pharmacological uses, and its nutritional value are discussed, as well as the potential of the species as a source of new and different chemical scaffolds. Very important in the past, appreciated in our time and extremely promising in the future, F. carica represents an interesting example of healthy foods and bioproducts.

Biophysical characteristics of successful oilseed embryo cryoprotection and cryopreservation using vacuum infiltration vitrification: an innovation in plant cell preservation.

Heterogeneity in morphology, physiology and cellular chemistry of plant tissues can compromise successful cryoprotection and cryopreservation. Cryoprotection is a function of exposure time × temperature × permeability for the chosen protectant and diffusion pathway length, as determined by specimen geometry, to provide sufficient dehydration whilst avoiding excessive chemical toxicity. We have developed an innovative method of vacuum infiltration vitrification (VIV) at 381 mm (15 in) Hg (50 kPa) that ensures the rapid (5 min), uniform permeation of Plant Vitrification Solution 2 (PVS2) cryoprotectant into plant embryos and their successful cryopreservation, as judged by regrowth in vitro. This method was validated on zygotic embryos/embryonic axes of three species (Carica papaya, Passiflora edulis and Laurus nobilis) up to 1.6 mg dry mass and 5.6 mm in length, with varying physiology (desiccation tolerances) and 80 °C variation in lipid thermal profiles, i.e., visco-elasticity properties, as determined by differential scanning calorimetry. Comparisons between the melting features of cryoprotected embryos and embryo regrowth indicated an optimal internal PVS2 concentration of about 60% of full strength. The physiological vigour of surviving embryos was directly related to the proportion of survivors. Compared with conventional vitrification, VIV-cryopreservation offered a ∼ 10-fold reduction in PVS2 exposure times, higher embryo viability and regrowth and greater effectiveness at two pre-treatment temperatures (0 °C and 25 °C). VIV-cryopreservation may form the basis of a generic, high throughput technology for the ex situ conservation of plant genetic resources, aiding food security and protection of species from diverse habitats and at risk of extinction.

Effects of nitric oxide treatment on the cell wall softening related enzymes and several hormones of papaya fruit during storage.

Papaya fruits (Carica papaya L. cv 'Sui you 2') harvested with < 5% yellow surface at the blossom end were fumigated with 60 microL/L of nitric oxide for 3 h and then stored at 20 degrees C with 85% relative humility for 20 days. The effects of nitric oxide treatment on ethylene production rate, the activities of cell wall softening related enzymes including polygalacturonase, pectin methyl esterase, pectate lyase and cellulase and the levels of hormones including indole acetic acid, abscisic acid, gibberellin and zeatin riboside were examined. The results showed that papaya fruits treated with nitric oxide had a significantly lower rate of ethylene production and a lesser loss of firmness during storage. A decrease in polygalacturonase, pectin methyl esterase, pectate lyase and cellulase activities was observed in nitric oxide treated fruit. In addition, the contents of indole acetic acid, abscisic acid and zeatin riboside were reduced in nitric oxide treated fruit, but no significant reduction in the level of gibberellin was found. These results indicate that nitric oxide treatment can effectively delay the softening and ripening of papaya fruit, likely via the regulation of cell wall softening related enzymes and certain hormones.

Stevia rebaudiana Bertoni as a natural antioxidant/antimicrobial for high pressure processed fruit extract: processing parameter optimization.

Response surface methodology was used to evaluate the optimal high pressure processing treatment (300-500 MPa, 5-15 min) combined with Stevia rebaudiana (Stevia) addition (0-2.5% (w/v)) to guarantee food safety while maintaining maximum retention of nutritional properties. A fruit extract matrix was selected and Listeria monocytogenes inactivation was followed from the food safety point of view while polyphenoloxidase (PPO) and peroxidase (POD) activities, total phenolic content (TPC) and antioxidant capacity (TEAC and ORAC) were studied from the food quality point of view. A combination of treatments achieved higher levels of inactivation of L. monocytogenes and of the oxidative enzymes, succeeding in completely inactivating POD and also increasing the levels of TPC, TEAC and ORAC. A treatment of 453 MPa for 5 min with a 2.5% (w/v) of Stevia succeeded in inactivating over 5 log cycles of L. monocytogenes and maximizing inactivation of PPO and POD, with the greatest retention of bioactive components.

Efeito fungitóxico do óleo essencial de aroeira da praia (Schinus terebinthifolius RADDI) sobre Colletotrichum gloeosporioides / Fungitoxic effect of essential oil from aroeira (Schinus terebinthifolius RADDI) on Colletotrichum gloeosporioides

Neste trabalho foi avaliado o efeito do óleo essencial do fruto de Schinus terebinthifolius sobre o crescimento micelial do fungo Colletotrichum gloeosporioides in vitro, e no desenvolvimento da antracnose no período de pós-colheita em mamões. As diferentes concentrações de óleo foram diluídas em Tween 80 a 8%. No experimento in vitro foram preparados meios de cultura BDA nas concentrações de 0,05; 0,10; 0,25 e 0,50% do óleo essencial. O controle negativo foi realizado apenas com meio BDA e o controle solvente com meio BDA e Tween 80 a 8%. A inibição do crescimento do fungo foi diretamente proporcional à quantidade do óleo e a maior inibição encontrada foi de 79,07% na concentração de óleo de 0,50%. No experimento in vivo os frutos do mamoeiro foram inoculados com o fungo em quatro tratamentos: com biofilme; com biofilme mais 0,50% do óleo; com fungicida Prochloraz e frutos controle. Embora o tratamento com óleo tenha sido eficiente contra o fungo, não foi indicado comercialmente, pois apresentou valores elevados de perda de massa fresca, de firmeza, e também sintomas de fitotoxidade. O óleo tem propriedade antifúngica contra C. gloeosporioides in vitro e in vivo, contudo, não é recomendado para o mamão em função da fitotoxidez.

This study evaluated the effect of essential oil from Schinus terebinthifolius fruit on the mycelial growth of the fungus Colletotrichum gloeosporioides in vitro and on the anthracnose development during the postharvest period of papaya fruits. The different oil concentrations were diluted in 8% Tween 80. For the in vitro experiment, PDA culture media were prepared at the concentrations of 0.05, 0.10, 0.25 and 0.50% essential oil. Negative control was prepared with PDA medium alone, while solvent control was prepared with PDA medium and 8% Tween 80. Fungal growth inhibition was directly proportional to the oil amount and the greatest inhibition was 79.07% at 0.50% oil concentration. For the in vivo experiment, papaya fruits were inoculated with the fungus in four treatments: with biofilm, with biofilm plus 0.50% oil, with the fungicide Prochloraz and control fruits. Although treatment with oil was efficient against the fungus, it was not commercially recommended since it presented high values of loss of fresh mass and firmness, as well as phytotoxicity symptoms. The oil has antifungal property against C. gloeosporioides both in vitro and in vivo; however, it is not recommended for papaya fruits due to its phytotoxicity.

Influence of host fruit and conspecifics on the release of the sex pheromone By Toxotrypana curvicauda males (Diptera: Tephritidae).

The release of tephritid sex pheromones depends significantly on the age of the male, the social context (presence of conspecifics) and chemical context (host volatiles). In this study, the influence of host fruit and conspecific (males and females) on the emission of the pheromonal compound 2-methyl-6-vinylpyrazine (2,6 mvp) by Toxotrypana curvicauda (Gerstaecker) males was investigated under laboratory conditions. Males were divided into one control group (nonexposed to treatment) and five experimental groups were placed 1 hr before volatile collection with: 1) host fruit (unripe papaya), 2) two females, 3) two males, 4) host fruit plus two females, and 5) host fruit plus two males. The volatiles were sampled by means of solid phase microextraction and analyzed with gas chromatography-mass spectrometry, with a focus on 2,6 mvp. Males emitted volatiles from the first day after adult emergence. The maximum peak and the temporal pattern of 2,6 mvp release were modified by the presence of host fruit and conspecific males or females. On day 5, males in the presence of fruit maintained a constant release of 2,6 mvp while the presence of conspecific caused a decrease of pheromone release. The release of 2,6 mvp was increased significantly in males exposed to both types of stimuli simultaneously. The stimuli (fruit and conspecifics) modified the release of 2,6 mvp, however the effect depended on male age.

The in vitro secretome of Mycosphaerella fijiensis induces cell death in banana leaves.

The hemibiotrophic filamentous fungus Mycosphaerella fijiensis causes the banana foliar disease known as black Sigatoka, responsible for major worldwide losses in the banana fruit industry. In this work the in vitro secretome of M. fijiensis was characterized. Native and denaturant polyacrylamide gel protease assays showed the M. fijiensis secretome contains protease activity capable of degrading gelatin. Necrotic lesions on leaves were produced by application of the in vitro secretome to the surface of one black Sigatoka-resistant banana wild species, one susceptible cultivar and the non-host plant Carica papaya. To distinguish if necrosis by the secretome is produced by phytotoxins or proteins, the latter ones were precipitated with ammonium sulfate and applied in native or denatured forms onto leaves of the same three plant species. Proteins applied in both preparations were able to produce necrotic lesions. Application of Pronase, a commercial bacterial protease suggested that the necrosis was, at least in part, caused by protease activity from the M. fijiensis secretome. The ability to cause necrotic lesions between M. fijiensis secreted- and ammonium sulfate-precipitated proteins, and purified lipophilic or hydrophilic phytotoxins, was compared. The results suggested that leaf necrosis arises from the combined action of non-host specific hydrolytic activities from the secreted proteins and the action of phytotoxins. This is the first characterization of the M. fijiensis protein secretome produced in vitro but, more importantly, it is also the first time the M. fijiensis secretome has been shown to contain virulence factors capable of causing necrosis to its natural host.

Elimination of phenols, ammonia and cyanide in wash water from biomass gasification, and nitrogen recycling using planted trickling filters.

Trickling filters were used to treat wash water from a wood gasifier. This wash water contained toxic substances such as ammonium, cyanide, phenols, and PAH. The goal was to develop a system that degraded toxic substances, and achieved full nitrification of ammonia. A 1 kW model wood gasifier plant delivered wash water for the experiments, which was standardised to a conductivity of 3 mS/cm by dilution. Toxicity was assessed by bacterial luminescence detection, germination test with cress (Lepidium sativum), and pot plants cultivated in a hydroponic setup irrigated continuously with the wastewater. Treatment experiments were done in both planted and unplanted trickling filters. Plant yield was similar to conventional hydroponic production systems. The trickling filters achieved complete detoxification of phenol, PAH and cyanide as well as full nitrification. The specific elimination rates were 100 g m(-3) Leca d(-1) for phenols and 90 g m(-3) Leca d(-1) for ammonium in planted systems. In unplanted trickling filters circulated for 63 h, phenol concentration decreased from 83.5 mg/L to 2.5 mg/L and cyanide concentration from 0.32 mg/L to 0.02 mg/L. PAH concentrations were reduced from 3,050 microg/L to 0.89 microg/L within 68 days. The assays demonstrated the feasibility of using the technique to construct a treatment system in a partially closed circulation for gasifier wash water. The principal advantage is to convert toxic effluents from biomass gasifiers into a non-toxic, nitrogen-rich fertiliser water, enabling subsequent use in plant production and thus income generation. However, the questions of long-term performance and possible accumulation of phenols and heavy metals in the produce still have to be studied.

Papaya (Carica papaya L.).

Transgenic papaya plants were initially obtained using particle bombardment, a method having poor efficiency in producing intact, single-copy insertion of transgenes. Single-copy gene insertion was improved using Agrobacterium tumefaciens. With progress being made in genome sequencing and gene discovery, there is a need for more efficient methods of transformation in order to study the function of these genes. We describe a protocol for Agrobacterium-mediated transformation using carborundum-wounded papaya embryogenic calli. This method should lead to high-throughput transformation, which on average produced at least one plant that was positive in polymerase chain reaction (PCR), histochemical staining, or by Southern blot hybridization from 10 to 20% of the callus clusters that had been co-cultivated with Agrobacterium. Plants regenerated from the callus clusters in 9 to 13 mo.