Endophytic species of Colletotrichum associated with cashew tree in northeastern Brazil.

Anthracnose caused by Colletotrichum is the most severe and widely occurring cashew disease in Brazil. Colletotrichum species are commonly found as pathogens, endophytes and occasionally as saprophytes in a wide range of hosts. The endophytic species associated with cashew trees are poorly studied. In this study, we report the Colletotrichum endophytic species associated with cashew trees in two locations in the state of Pernambuco, their prevalence in different plant organs (leaves, veins, branches and inflorescences), and compare the species in terms of pathogenicity and aggressiveness using different inoculation methods (wounded × unwounded). Six species of Colletotrichum were identified according to multilocus phylogenetic analyses, including Colletotrichum asianum, Colletotrichum chrysophilum, Colletotrichum karsti, Colletotrichum siamense, Colletotrichum theobromicola, and Colletotrichum tropicale. There were differences in the percentage of isolation in relation to the prevalence of colonized tissues and collection locations. C. tropicale was the prevalent species in both geographic areas and plant tissues collected, with no pattern of distribution of species between areas and plant tissues. All isolates were pathogenic in injured tissues of cashew plants. The best method to test the pathogenicity of Colletotrichum species was utilizing the combination of leaves + presence of wounds + conidial suspension, as it better represents the natural infection process. C. siamense was the most aggressive species.

Survey of Salmonella in raw tree nuts at retail in the United States.

The objective of this survey was to estimate the prevalence, contamination level, and genetic diversity of Salmonella in selected raw, shelled tree nuts (Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios, and walnuts) at retail markets in the United States. A total of 3,374 samples of eight tree nuts were collected from different types of retail stores and markets nationwide between September 2015 and March 2017. These samples (375 g) were analyzed using a modified FDA's BAM Salmonella culture method. Of the 3,374 samples, 15 (0.44%) (95% confidence interval [CI] [0.25, 0.73]) were culturally confirmed as containing Salmonella; 17 isolates were obtained. Among these isolates, there were 11 serotypes. Salmonella was not detected in Brazil nuts (296), hazelnuts (487), pecans (510), pine nuts (500), and walnuts (498). Salmonella prevalence estimates in cashews (510), macadamia (278), and pistachios (295) were 0.20% (95% CI [<0.01, 1.09]), 2.52% (95% CI [1.02, 5.12]), and 2.37% (95% CI [0.96, 4.83]), respectively. The rates of Salmonella isolation from major/big-chain supermarkets (1381), small-chain supermarkets (328), discount/variety/drug stores (1329), and online (336) were 0.29% (95% CI [0.08, 0.74]), 0.30% (95% CI [0.01, 1.69]), 0.45% (95% CI [0.17, 0.98]), and 1.19% (95% CI [0.33, 3.02]), respectively. Salmonella prevalence in organic (530) and conventional (2,844) nuts was not different statistically (P = 0.0601). Of the enumerated samples (15), 80% had Salmonella levels ≤0.0092 most probable number (MPN)/g. The highest contamination level observed was 0.75 MPN/g. The prevalence and contamination levels of Salmonella in the tree nuts analyzed were generally comparable to previous reports. Pulsed-field gel electrophoresis, serotype, and sequencing data all demonstrated that Salmonella population in nuts is very diverse genetically. PRACTICAL APPLICATION: The prevalence, contamination level, and genetic diversity of Salmonella in eight types of tree nuts (3,374 samples collected nationwide) revealed in this survey could help the development of mitigation strategies to reduce public health risks associated with consumption of these nuts.

Multifunctional potential of endophytic bacteria from Anacardium othonianum Rizzini in promoting in vitro and ex vitro plant growth.

Anacardium othonianum Rizzini, a cashew tree native to the Brazilian Cerrado, is economically important due to its applications in the food, chemical and pharmaceutical industries. However, A. othonianum yields a crop with low productivity due to a number of factors, such as nutritionally poor soils, drought and losses due to pests and diseases. Brazil is one of the nine largest cashew nut producers worldwide, and sustainable technologies are needed to increase the productivity of this crop. In this context, the use of endophytic microorganisms could promote plant growth and provide protection against phytopathogens. In this study, the isolation of the root endophytic community of A. othonianum led to the characterization of 22 distinct bacterial strains with multifunctional traits for plant growth promotion. The results of in vitro assays to assess auxin synthesis, phosphate solubilization, phosphatase and siderophore production and biocontrol against Fusarium oxysporum led to the selection of Acinetobacter lwoffii Bac109 and Pantoea agglomerans Bac131 as the most promising strains. The reinoculation of the Bac109 and Bac131 strains onto A. othonianum seeds showed that the treatment containing a mixture of these strains was the most effective in promoting increases in the biometric parameters of early plant growth. Thus, this study highlights the biotechnological potential of a consortium of A. lwoffii Bac109 and P. agglomerans Bac131 for future applications in sustainable cashew cultivation.

Vinegar from Anacardium othonianum Rizzini using submerged fermentation.

BACKGROUND: Anacardium othonianum Rizzini is a native Cerrado fruit, recently described in the literature. Its use is restricted to its native region and there is a lack of studies regarding production of vinegar from the pulp. This work aims to investigate the production of A. othonianum Rizzini vinegar using submerged fermentation. RESULTS: The density, alcohol content, proximal composition, pH, color coordinates, and chromatographic profile of the volatile compounds were analyzed in the slurry, fermented juice, and vinegar produced from the corpulent parts of A. othonianum Rizz. Sensory acceptance and willingness to pay were also assessed with vinegar at 4% and 6% of total acidity. The results indicated compliance with European legislation and the presence of volatile compounds such as carbon dioxide, acetic acid, ethanol, and acetaldehyde in the analyzed vinegars. Our results indicate the potential of vinegar production from A. othonianum, with 74% and 86% willingness to pay. CONCLUSIONS: The process of transformation of the fruit pulp into new products can contribute to fruit valorization and consequent preservation of the plant in the Cerrado biome. To the best of our knowledge, this is the first report of volatile compounds and minerals in A. othonianum Rizz. slurry. Our observations can be used as a basis for future studies regarding the preparation of vinegars from this species and for investigating their application in cooking and guiding consumer perception. © 2020 Society of Chemical Industry.

A phylogeny for the plant pathogen Piptoporellus baudonii using a multigene data set.

Piptoporellus baudonii is proposed as a new combination for Laetiporus baudonii in the Polyporales (Basidiomycota) based on morphological and molecular features. This parasitic macrofungus attacks cashew trees, Eucalyptus, cassava, Tectona, and some indigenous trees in southern regions of Tanzania and poses a serious threat to agroforestry and livelihood conditions in the area. Phylogenetic trees were produced from partial sequences of three rDNA gene regions and a portion of translation elongation factor 1-alpha (TEF1) gene of Laetiporus baudonii for comparisons with samples from the antrodia clade. Our results reveal a strongly supported group of L. baudonii with Piptoporellus in Fomitopsidaceae. Piptoporellus baudonii shares many morphological features with other members of Piptoporellus but differs from them in having broadly ellipsoid or rarely ovoid basidiospores. Both morphological and phylogenetic evidence justify the placement of L. baudonii in Piptoporellus together with the three other known species in the genus.

Detection of Colletotrichum gloeosporioides in native cashew species in Brazil.

The savanna cashew (Anacardium humile A. St. Hil.) nut is a native shrub species distributed in the savanna biome of Central Western Brazil. This species is similar to the cashew (Anacardium occidentale L.) nut, and its edible pseudo-fruits have been used for various purposes. However, A. humile is at risk of extinction. One reason for this is the phytosanitary issues. Therefore, phytosanitary studies of the serious plant diseases caused by fungal pathogens are necessary. The aim of this study was to survey the savanna cashew nut (Anacardium humile) in Central Western Brazil from August 2013 to October 2014. Serious damage caused by anthracnose to the plant's leaves and fruits was consistently detected. The pathogen fungal species was Colletotrichum gloeosporioides. It was identified by morphological characteristics and molecular analyses. Additional experiments showed evidence that C. gloeosporioides can be transmitted by cashew seeds. We observed anthracnose symptoms on the surfaces of the floral peduncle, leaves, and peduncle of the A. humile cashew nuts. The genomic DNA sequences of the selected strains for molecular characterization have had 99% identity with the analogous sequences of C. gloeosporioides. Naturally occurring C. gloeosporioides infection of the leaves of A. humile was low (20.3%) compared with that of the nuts (79.7%). Our results can be used to design strategies to prevent the introduction and establishment of C. gloeosporioides in new areas. They can also be helpful in monitoring programs in areas with a current occurrence of C. gloeosporioides. Finally, these results can be used in future research plans of C. gloeosporioides infection management.

A polyphasic method for the identification of aflatoxigenic Aspergilla from cashew nuts.

The invasion of food by toxigenic fungi is a threat to public health. This study aimed at enumerating the microbial profile, detection of aflatoxin producing genes and quantification of the levels of aflatoxin contamination of cashew nuts meant for human consumption. A polyphasic method of analysis using newly formulated ß-Cyclodextrin Neutral Red Desiccated coconut agar (ß-CDNRDCA) and Yeast Extract Sucrose agar (YES) with Thin Layer Chromatography (TLC), Polymerase Chain Reaction (PCR) and High Performance Liquid Chromatographic (HPLC) method was adopted in determining the aflatoxigenic potential of the isolates, the presence of aflatoxin biosynthetic gene (aflM, aflD, aflR, aflJ omt-A) and estimation of the total aflatoxin content of the nuts. The fungal counts ranged from 2.0 to 2.4 log10cfu/g and sixty-three fungal isolates belonging to 18 genera and 34 species were isolated. The Aspergillus spp. were the most frequently isolated (50.79%) while Trichoderma spp. (1.59%) were the least. and fluorescence production was enhanced on the newly formulated ß-CDNRDCA by the aflatoxigenic species. The aflD gene was amplified in all the isolates while aflM, aflR and aflJ gene were each amplified in 77.77% of the isolates and omt-A gene in 70.37%. The aflatoxin content of the nuts ranged from 0.03 to 0.77 µg/kg and were below the 4 µg/kg EU recommended limit for total aflatoxins. The present work confirms that a single method of analysis may not be sufficient to screen for the presence of aflatoxins in foods, as with a combination of different methods.

Why species delimitation matters for fungal ecology: Colletotrichum diversity on wild and cultivated cashew in Brazil.

Anthracnose is one of the most important plant diseases globally, occurring on a wide range of cultivated and wild host species. This study aimed to identify the Colletotrichum species associated with cashew anthracnose in Brazil, determine their phylogenetic relationships and geographical distribution, and provide some insight into the factors that may be influencing community composition. Colletotrichum isolates collected from symptomatic leaves, stems, inflorescences, and fruit of cultivated and wild cashew, across four Brazilian biomes, were identified as Colletotrichum chrysophilum, Colletotrichum fragariae, Colletotrichum fructicola, Colletotrichum gloeosporioides sensu stricto, Colletotrichum queenslandicum, Colletotrichum siamense and Colletotrichum tropicale. Colletotrichum siamense was the most dominant species. The greatest species richness was associated with cultivated cashew; leaves harbored more species than the other organs; the Atlantic Forest encompassed more species than the other biomes; and Pernambuco was the most species-rich location. However, accounting for the relative abundance of Colletotrichum species and differences in sample size across strata, the interpretation of which community is most diverse depends on how species are delimited. The present study provides valuable information about the Colletotrichum/cashew pathosystem, sheds light on the causal agents identification,and highlights the impact that species delimitation can have on ecological studies of fungi.

Taxonomic Repositioning of Xanthomonas campestris pv. viticola (Nayudu 1972) Dye 1978 as Xanthomonas citri pv. viticola (Nayudu 1972) Dye 1978 comb. nov. and Emendation of the Description of Xanthomonas citri pv. anacardii to Include Pigmented Isolates Pathogenic to Cashew Plant.

Grapevine bacterial canker, which is caused by Xanthomonas campestris pv. viticola, is one of the most important grapevine diseases in the northeastern region of Brazil. This disease causes severe damage and represents a high potential risk to the development of Brazilian viticulture. In turn, pigmented isolates pathogenic to cashew plant, making cashew fruit unfit for sale, also have been detected in Northeastern Brazil. Given that the taxonomic position of these bacteria is unclear, the multilocus sequence analysis (MLSA) technique, average nucleotide identity (ANI) values and tetranucleotide frequency correlation coefficients (TETRA) were used to analyze their phylogenetic relationship in relation to other Xanthomonas species. X. campestris pv. viticola was closely related to X. citri pv. mangiferaeindicae (repetitive-polymerase chain reaction [rep-PCR], MLSA, and ANI) and X. citri subsp. citri (MLSA and ANI). Pigmented isolates pathogenic to cashew plant were closely related to X. citri pv. anacardii (rep-PCR, MLSA, ANI, and TETRA). The results obtained in this study support the emendation of the description of X. citri pv. anacardii to include pigmented isolates of Xanthomonas pathogenic to cashew plant. In addition, the reclassification of X. campestris pv. viticola as X. citri pv. viticola comb. nov. is suggested.

Inactivation of Salmonella and Surrogate Bacteria on Cashews and Macadamia Nuts Exposed to Commercial Propylene Oxide Processing Conditions.

Propylene oxide (PPO), a chemical fumigant, has been validated to reduce Salmonella on bulk almonds but has not been evaluated for other tree nuts. There is a need to identify nonpathogenic surrogate microorganisms whose inactivation is comparable to that of Salmonella to assure effective PPO processing parameters in different packaging configurations without introducing Salmonella into the pasteurization facility. The objective of this research was to compare the reduction of Salmonella and three potential surrogate bacterial strains, Enterococcus faecium ATCC 8459, Pediococcus acidilactici ATCC 8042, or Staphylococcus carnosus ATCC 51365, on cashews and macadamia nuts processed by using PPO. Whole cashews and macadamia nuts were coinoculated with a five-strain cocktail of Salmonella and one surrogate, dried to the original water activity of 0.44 to 0.51 before being packaged in woven polypropylene bags (2.3 kg), and shipped overnight in Styrofoam containers under ambient conditions to a commercial facility for PPO treatment. Salmonella and surrogates were recovered by vigorous shaking in phosphate buffer (1:1, m/v), serial diluted, and plated onto tryptic soy agar with an overlay of xylose lysine Tergitol 4 for Salmonella or mannitol salt agar or bile esculin azide agar for each surrogate. The mean log reductions of Salmonella and each surrogate ( n = 18), within a sample and among all trials (three independent), were compared by using a matched pairs t test. Reduction in log CFU per gram of Salmonella was significantly greater than that of E. faecium on both macadamia nuts (7.3 ± 0.19 versus 6.4 ± 0.31) and cashews (5.4 ± 0.15 versus 5.1 ± 0.25) and significantly greater than P. acidilactici on both nuts (7.8 ± 0.22 versus 6.3 ± 0.33 on macadamia nuts and 4.9 ± 0.22 versus 4.1 ± 0.25 on cashews). Reduction of S. carnosus exceeded that of Salmonella. E. faecium and P. acidilactici may be considered as surrogates for Salmonella on whole macadamia nuts and cashews processed by using PPO.

Chemometric evaluation of the volatile profile of probiotic melon and probiotic cashew juice.

The aim of this study was to evaluate the influence of the lactic acid fermentation on volatile compounds of melon and cashew apple juices. The effect of the fermentation processing on the volatile profile of probiotic juices was assessed by HS-SPME/GC-MS coupled to chemometrics with 67.9% and 81.0% of the variance in the first principal component for melon and cashew juices, respectively. The Lactobacillus casei fermentation imparted a reduction of ethyl butanoate, ethyl-2-methylbutirate, and ethyl hexanoate for melon juice; and of ethyl acetate, ethyl-2-methyl butanoate, ethyl crotonate, ethyl isovalerate, benzaldehyde, and ethyl hexanoate for cashew juice. Measurements of the stability of these compounds and the formation of the component 3-methyl-2-butenyl in melon juice may be used as a volatile marker to follow the juice fermentation. These findings suggested that even though it is not a dairy product the lactic acid fermentation of fruits developed a volatile profile combining the fruit and lactic acid fermentation volatiles with mildly formation or degradation of aroma compounds.

Analysis of phylogeny, distribution, and pathogenicity of Botryosphaeriaceae species associated with gummosis of Anacardium in Brazil, with a new species of Lasiodiplodia.

Netto, M. S. B., Lima, W. G., Correia, K. C., da Silva, C. F. B., Thon, M., Martins, R. B., Miller, R. N. G., Michereff, S. J., and Câmara, M. P. S. 2016. Analysis of phylogeny, distribution, and pathogenicity of Botryosphaeriaceae species associated with gummosis of Anacardium in Brazil, with a new species of Lasiodiplodia. We identified Botryosphaeriaceae species associated with gummosis on Anacardium in Brazil. Isolates were sampled and identified on the basis morphology and phylogeny, through analysis of a partial translation elongation factor 1-α sequence, ribosomal DNA internal transcribed spacers, and ß-tubulin gene sequence. Ten taxa were identified, namely, Lasiodiplodia brasiliense, L. euphorbicola, L. gonubiensis, L. iraniensis, L. jatrophicola, L. gravistriata sp. nov., L. pseudotheobromae, L. theobromae, Neofusicoccum batangarum, and Pseudofusicoccum stromaticum. Lasiodiplodia theobromae has been previously reported in cashew and is the most prevalent species observed. All the other species are reported here for the first time on this host. All species of Botryosphaeriaceae were pathogenic on detached green cashew shoots. Differences in aggressiveness were observed among the species, with N. batangarum, L. iraniensis, L. jatrophicola, and L. gravistriata characterized as the most aggressive species, whilst L. euphorbicola and L. pseudotheobromae were identified as the least aggressive.

Potential prebiotic properties of cashew apple (Anacardium occidentale L.) agro-industrial byproduct on Lactobacillus species.

BACKGROUND: The prebiotic effects of a cashew apple (Anacardium occidentale L.) agro-industrial byproduct powder (CAP) on different potentially probiotic Lactobacillus strains, namely Lactobacillus acidophilus LA-05, Lactobacillus casei L-26 and Lactobacillus paracasei L-10, were assessed using in vitro experimental models. Accordingly, the growth of the Lactobacillus strains when cultivated in a broth containing CAP (20 or 30 g L-1 ), glucose (20 g L-1 ) or fructooligosaccharides (FOS) (20 g L-1 ) was monitored over 48 h; the prebiotic activity scores of CAP were determined; and the changes in pH values, production of organic acids and consumption of sugars in growth media were verified. RESULTS: During the 48-h cultivation, similar viable cell counts were observed for the Lactobacillus strains grown in the different media tested. The CAP presented positive prebiotic activity scores toward all the tested Lactobacillus strains, indicating a desirable selective fermentable activity relative to enteric organisms. The cultivation of the Lactobacillus strains in broth containing glucose, FOS or CAP resulted in high viable cell counts, a decreased pH, the production of organic acids and the consumption of sugars over time, revealing intense bacterial metabolic activity. CONCLUSION: The CAP exerts potential prebiotic effects on different potentially probiotic Lactobacillus strains and should be an added-value ingredient for the food industry. © 2017 Society of Chemical Industry.

Diversity in Secondary Metabolites Including Mycotoxins from Strains of Aspergillus Section Nigri Isolated from Raw Cashew Nuts from Benin, West Africa.

In a previous study, raw cashew kernels were assayed for the fungal contamination focusing on strains belonging to the genus Aspergillus and on aflatoxins producers. These samples showed high contamination with Aspergillus section Nigri species and absence of aflatoxins. To investigate the diversity of secondary metabolites, including mycotoxins, the species of A. section Nigri may produce and thus threaten to contaminate the raw cashew kernels, 150 strains were isolated from cashew samples and assayed for their production of secondary metabolites using liquid chromatography high resolution mass spectrometry (LC-HRMS). Seven species of black Aspergilli were isolated based on morphological and chemical identification: A. tubingensis (44%), A. niger (32%), A. brasiliensis (10%), A. carbonarius (8.7%), A. luchuensis (2.7%), A. aculeatus (2%) and A. aculeatinus (0.7%). From these, 45 metabolites and their isomers were identified. Aurasperone and pyranonigrin A, produced by all species excluding A. aculeatus and A. aculeatinus, were most prevalent and were encountered in 146 (97.3%) and 145 (95.7%) isolates, respectively. Three mycotoxins groups were detected: fumonisins (B2 and B4) (2.7%) ochratoxin A (13.3%), and secalonic acids (2%), indicating that these mycotoxins could occur in raw cashew nuts. Thirty strains of black Aspergilli were randomly sampled for verification of species identity based on sequences of ß-tubulin and calmodulin genes. Among them, 27 isolates were positive to the primers used and 11 were identified as A. niger, 7 as A. tubingensis, 6 as A. carbonarius, 2 as A. luchuensis and 1 as A. welwitschiae confirming the species names as based on morphology and chemical features. These strains clustered in 5 clades in A. section Nigri. Chemical profile clustering also showed also 5 groups confirming the species specific metabolites production.

The efficacy of Mentha arvensis L. and M. piperita L. essential oils in reducing pathogenic bacteria and maintaining quality characteristics in cashew, guava, mango, and pineapple juices.

This study evaluated the ability of the essential oil from Mentha arvensis L. (MAEO) and M. piperita L. (MPEO) to induce ≥5-log reductions in counts (CFU/mL) of E. coli, L. monocytogenes, and Salmonella enterica serovar Enteritidis in Brain-Heart Infusion broth (BHIB) and cashew, guava, mango, and pineapple juices during refrigerated storage (4±0.5°C). The effects of the incorporation of these essential oils on some physicochemical and sensory parameters of juices were also evaluated. The incorporation of 5, 2.5, 1.25, or 0.625µL/mL of MAEO in BHIB caused a ≥5-log reduction in counts of E. coli and Salmonella Enteritidis after 24h of storage; but only 5µL/mL was able to cause the same reduction in counts of L.monocytogenes. The incorporation of 10µL/mL of MPEO in BHIB caused a ≥5-log reduction in counts of E. coli, Salmonella Enteritidis, and L. monocytogenes after 24h of storage; smaller reductions were observed in BHIB containing 5, 2.5, and 1.25µL/mL of MPEO. Similar reductions were observed when the MAEO or MPEO was incorporated at the same concentrations in mango juice. The incorporation of MAEO or MPEO at all tested concentrations in cashew, guava, and pineapple juices resulted in a ≥5-log reduction in pathogen counts within 1h. The incorporation of MAEO and MPEO (0.625 and 1.25µL/mL, respectively) in fruit juices did not induce alterations in °Brix, pH, and acidity, but negatively affected the taste, aftertaste, and overall acceptance. The use of MAEO or MPEO at low concentrations could constitute an interesting tool to achieve the required 5-log reduction of pathogenic bacteria in cashew, guava, mango, and pineapple fruit juices. However, new methods combining the use of MAEO or MPEO with other technologies are necessary to reduce their negative impacts on specific sensory properties of these juices.

Proteomic analysis of responsive stem proteins of resistant and susceptible cashew plants after Lasiodiplodia theobromae infection.

Gummosis is an aggressive disease caused by the necrotrophic fungus Lasiodiplodia theobromae (Pat.) Griffon & Maubl that threatens commercial cashew orchads in Brazil. To study the molecular mechanisms involved in the cashew response to L. theobromae, a proteomic analysis of stems from the commercial cashew clone BRS 226 (resistant) was conducted at early times post-artificial infection. In addition, changes in the stem proteome profiles of gummosis resistant and susceptible cashew plants grown under field condition and naturally exposed to pathogen were also compared. After two-dimensional gel electrophoresis (2D-PAGE), 73 proteins showed statistically significant differences in spot abundance. Of these, 31 spots were identified in BRS 226 stems compared with mock-inoculated controls and 32 in stems collected from field-grown resistant and susceptible cashew plants. L. theobromae-responsive proteins were mainly involved in energy metabolism pathways, stress and defense, cell signaling and protein metabolism indicating modulation of various cellular functions upon fungal infection. As stress-inducing factors seem to be important for susceptibility to disease, the change in the abundance relative these proteins may possibly indicate an attempt to maintain cellular homeostasis, as resistance determinant factor, related with a possible role in the regulation of oxidative burst. These findings provide the first information about the cellular mechanisms acting in the Anacardium occidentale genotypes associated with the pathophysiological state of infection with L. theobromae. BIOLOGICAL SIGNIFICANCE: Gummosis caused by Lasiodiplodia theobromae, a necrotrophic fungus, is the major disease of cashew plants in the semi-arid conditions of northeastern Brazil. Although various studies were carried out on this pathosystem, there is no information available on the molecular mechanisms of plant defense related to the incompatible interaction of cashew with L. theobromae. Therefore, this original study comprises a differential proteomic analysis of cashew stems from: (i) resistant dwarf clone BRS 226 mock-inoculated (control) and artificially inoculated with L. theobromae; and (ii) cashew plants bearing resistant and susceptible traits to gummosis, originated from open pollination of BRS 226 in a commercial orchard with high disease incidence. The contribution of the reprogrammed proteins to molecular events triggered in cashew plants challenged by L. theobromae has a great relevance in the identification of the host candidate proteins linked to biological pathways that respond to L. theobromae infection. Furthermore this study may contribute to improve breeding programs aimed at selecting resistant/tolerant cashew clones toward this pathogen.

Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants.

Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation.

The ultrastructure of shelled and unshelled cashew nuts.

Cashew nuts have many attributes, including sensory, nutritional and health appeal, which contribute to their worldwide acceptance. We demonstrate details of the microstructure of shelled and unshelled cashew kernels with regard to pericarp and cotyledon organization. This study also provides evidence of the colonization of these kernels by filamentous fungi. Nuts were examined by scanning electron and confocal scanning laser microscopy. Staining with acridine orange was performed. A tight lignified palisade layer adjacent to the exocarp surface explains the hardness of the shell's pericarp. The mesocarp contains large secretory cavities that confer a spongy property to this tissue. Papillose cells, which are responsible for secreting CNSL (cashew nutshell liquid), were observed to cover the inner wall of these cavities. Lipid components are readily released from the parenchyma and appear as oil droplets. The outer surface of the shelled samples exhibited a dense Aspergillus infestation.

Ethanol production from cashew apple bagasse: improvement of enzymatic hydrolysis by microwave-assisted alkali pretreatment.

In this work, the potential of microwave-assisted alkali pretreatment in order to improve the rupture of the recalcitrant structures of the cashew able bagasse (CAB), lignocellulosic by-product in Brazil with no commercial value, is obtained from cashew apple process to juice production, was studied. First, biomass composition of CAB was determined, and the percentage of glucan and lignin was 20.54 ± 0.70% and 33.80 ± 1.30%, respectively. CAB content in terms of cellulose, hemicelluloses, and lignin, 19.21 ± 0.35%, 12.05 ± 0.37%, and 38.11 ± 0.08%, respectively, was also determined. Results showed that, after enzymatic hydrolysis, alkali concentration exerted influence on glucose formation, after pretreatment with 0.2 and 1.0 mo L(-1) of NaOH (372 ± 12 and 355 ± 37 mg g(glucan)(-1) ) when 2% (w/v) of cashew apple bagasse pretreated by microwave-assisted alkali pretreatment (CAB-M) was used. On the other hand, pretreatment time (15-30 min) and microwave power (600-900 W) exerted no significant effect on hydrolysis. On enzymatic hydrolysis step, improvement on solid percentage (16% w/v) and enzyme load (30 FPU g (CAB-M) (-1) ) increased glucose concentration to 15 g L(-1). The fermentation of the hydrolyzate by Saccharomyces cerevesiae resulted in ethanol concentration and productivity of 5.6 g L(-1) and 1.41 g L(-1) h(-1), respectively.

Colonization of cashew plants by Lasiodiplodia theobromae: microscopical features.

Lasiodiplodia theobromae is a phytopathogenic fungus causing gummosis, a threatening disease for cashew plants in Brazil. In an attempt to investigate the ultrastructural features of the pathogen colonization and its response to immunofluorescence labeling, light, confocal and electron microscope studies were conducted on different severity scale patterns of diseased plants. Lasiodiplodia-antisera was checked for cross reactivity against common cashew plants fungi. Optical microscopy analysis revealed a longitudinally sectioned hyphae located within the xylem vessels, showing an extensive hyphal development in the secondary xylem tissue. SEM images demonstrated that the fungus was found in some asymptomatic samples, particularly within the xylem vessels as confirmed by the optical images. Symptomatic sample images showed an extensive distribution of the fungus along the secondary xylem, within the vessels, infecting xylem parenchyma. A closer look in the secondary xylem parenchyma reveals a heavy and profuse invasion of the cells with a distinguishable cell wall disintegration and fully hyphae dispersal. There was no reactivity of Lasiodiplodia-antisera against mycelial extracts of Colletotrichum gloeosporioides, Phomopsis anardii and Pestalotiopsis guepinii. Following incubation of sections with the polyclonal antisera, the hyphae were intensely and regularly labeled. Rays, vessels and parenchyma cells were the preferred pathway for L. theobromae colonization. Artificial infection provides the information that the vascular cylinder is undoubtedly employed and used by the fungus for hyphae distribution. Immunofluorescence assay employed in situ was applied and the polyclonal antisera produced was able to recognize the fungus and proved to be a sensitive technique to detect it.