Studies on Pantoea citrea, the Causal Agent of Pink Disease of Pineapple

The causal agent of pink disease of pineapple has been identified as Pantoea citrea, a member of the Enterobacteriaceae. Comparative physiological and biochemical analyses demonstrated that P. citrea isolated from diseased pineapple fruit in the Philippines possesses features identical to those of an American Type Culture Collection type strain of P. citrea and not to those of P. ananas, P. herbicola (formerly Erwinia herbicola), and P. stewartii (formerly Erwinia stewartii). P. citrea induces the production of compounds in pineapple which become pink to reddish-brown upon cooking the fruit, pulp, or juice. This distinct colour is not induced by Escherichia coli, Agrobacterium tumefaciens, Burkholderia gladioli, Pseudomonas fluorescens, Gluconobacter oxydans, Acetobacter aceti, and Acinetobacter calcoaceticus. Like other well characterized bacteria pathogens, such as Pseudomonas syringae pv. phaseolicola, P. citrea elicits the hypersensitive response (HR) in tobacco. By contrast, G. oxydans and A. aceti that have been previously implicated as the causal agents of pink disease, do not elicit HR. Although the nature of the pink colour in pineapple produced by P. citrea has not been elucidated, the locus conferring this activity has been located on its chromosome. The pink colour can be restored in an avirulent, pink colour defective mutant strain, CMC6, by complementation in trans with a specific 3.8 kb genomic DNA fragment of P. citrea. This suggests that P. citrea contains the genetic elements that are required for pink disease.     

Characterization of Fusarium spp. associated with pineapple fruit rot and leaf spot in Peninsular Malaysia

Pineapple (Ananas comosus) is one the important fruit crops planted in Malaysia, and this study was conducted to determine Fusarium spp. associated with diseases of the fruit crop as Fusarium is prevalent in tropical countries. Our objective was to identify and characterize Fusarium spp. associated with pineapple fruit rot and leaf spot mainly found on the fruits and leaves in Peninsular Malaysia. Fusarium isolates (n = 108) associated with pineapple fruit rot and leaf spot were characterized by morphological, molecular and phylogenetic analyses, a mating study and pathogenicity testing. TEF‐1α sequence analysis identified Fusarium proliferatum, Fusarium verticillioides, Fusarium sacchari and Fusarium sp. Mating was successful only between tester strains of F. proliferatum and F. verticillioides. Sexual crosses with standard tester strains showed that 82 isolates of F. proliferatum produced fertile crosses with mating population D (Gibberella intermedia) and three isolates of F. verticillioides were fertile with the tester strain of mating population A (Gibberella moniliformis). All isolates were pathogenic, causing pineapple fruit rot and leaf spot, thus fulfilling Koch's postulates.     

Natural Endophytic Occurrence of Acetobacter diazotrophicus in Pineapple Plants

Abstract The presence of endophytic Acetobacter diazotrophicus was tested for pineapple plants (Ananas comosus [L.] Merr.) grown in the field. Diazotrophic bacteria were isolated from the inner tissues of surface sterilized roots, stems, and leaves of pineapple plants. Phenotypic tests permitted the selection of presumptive nitrogen-fixing A. diazotrophicus isolates. Restriction fragment length polymorphisms (RFLPs) of small subunit (SSU) rDNA using total DNA digested with endonuclease SphI and with endonuclease NcoI, hybridizations of RNA with an A. diazotrophicus large subunit (LSU) rRNA specific probe, as well as patterns in denaturing protein electrophoresis (SDS-PAGE) and multilocus enzyme tests allowed the identification of A. diazotrophicus isolates. High frequencies of isolation were obtained from propagative buds that had not been nitrogen-fertilized, and lower frequencies from 3-month-old plants that had been nitrogen-fertilized. No isolates were recovered from 5- to 7-month-old nitrogen-fertilized plants. All the A. diazotrophicus isolates recovered from pineapple plants belonged to the multilocus genotype which shows the most extensive distribution among all host species previously analyzed. Received: 16 March 1999; Accepted: 27 August 1999; Online Publication: 23 February 2000     

Genetic and biochemical characterization of the pathway in Pantoea citrea leading to pink disease of pineapple

Pink disease of pineapple, caused by Pantoea citrea, is characterized by a dark coloration on fruit slices after autoclaving. This coloration is initiated by the oxidation of glucose to gluconate, which is followed by further oxidation of gluconate to as yet unknown chromogenic compounds. To elucidate the biochemical pathway leading to pink disease, we generated six coloration-defective mutants of P. citrea that were still able to oxidize glucose into gluconate. Three mutants were found to be affected in genes involved in the biogenesis of c-type cytochromes, which are known for their role as specific electron acceptors linked to dehydrogenase activities. Three additional mutants were affected in different genes within an operon that probably encodes a 2-ketogluconate dehydrogenase protein. These six mutants were found to be unable to oxidize gluconate or 2-ketogluconate, resulting in an inability to produce the compound 2,5-diketogluconate (2,5-DKG). Thus, the production of 2,5-DKG by P. citrea appears to be responsible for the dark color characteristic of the pink disease of pineapple.     

Molecular characterization and pathogenicity of Colletotrichum sp. from guava

Guava (Psidium guajava) fruit is vulnerable to postharvest diseases, such as anthracnose. In the present study, molecular characterisation and pathogenicity of Colletotrichum associated with antharcnose disease of guava fruit were conducted. From anthracnose lesion of guava, 20 isolates were successfully recovered. Based on colony colours, conidia, appressoria and presence or absence of setae, and ITS regions and ß-tubulin gene sequences, the isolates were identified as Colletotrichum gloeosporioides. Phylogenetic analysis based on combined data-sets using neighbour-joining method showed that C. gloeosporioides isolates did not group with C. gloeosporioides epitype strain, and thus the isolates were referred to as C. gloeosporioides species complex or C. gloeosporioides sensu lato. Pathogenicity tests using wounded treatment showed that C. gloeosporioides isolates from guava were pathogenic causing anthracnose on the fruits. The present study showed that C. gloeosporioides sensu lato is the most common species causing antharcnose disease of guava fruit.     

Diversity of Fusarium species and mycotoxins contaminating pineapple

Pineapple (Ananas comosus var. comosus) is an important perennial crop in tropical and subtropical areas. It may be infected by various Fusarium species, contaminating the plant material with mycotoxins. The aim of this study was to evaluate Fusarium species variability among the genotypes isolated from pineapple fruits displaying fungal infection symptoms and to evaluate their mycotoxigenic abilities. Forty-four isolates of ten Fusarium species were obtained from pineapple fruit samples: F. ananatum, F. concentricum, F. fujikuroi, F. guttiforme, F. incarnatum, F. oxysporum, F. polyphialidicum, F. proliferatum, F. temperatum and F. verticillioides. Fumonisins B₁–B₃, beauvericin (BEA) and moniliformin (MON) contents were quantified by high-performance liquid chromatography (HPLC) in pineapple fruit tissue. Fumonisins are likely the most dangerous metabolites present in fruit samples (the maximum FB₁ content was 250 μg g^?1 in pineapple skin and 20 μg ml^?1 in juice fraction). In both fractions, BEA and MON were of minor significance. FUM1 and FUM8 genes were identified in F. fujikuroi, F. proliferatum, F. temperatum and F. verticillioides. Cyclic peptide synthase gene (esyn1 homologue) from the BEA biosynthetic pathway was identified in 40 isolates of eight species. Based on the gene-specific polymerase chain reaction (PCR) assays, none of the isolates tested were found to be able to produce trichothecenes or zearalenone.     

Nutritional requirements and biochemical activities of pineapple pink disease bacterial strains from Hawaii

Bacteria which cause pink disease of pineapple, identified on the basis of their nutritional and biochemical activities, were found to belong to three genera. These bacteria include the following species: Gluconobacter oxydans, Acetobacter aceti, and Erwinia herbicola. Several pink disease strains required one to three vitamins for growth. Both G. oxydans strains 303D and 180 required biotin, nicotinic acid, and pantothenic acid for growth; E. herbicola 189 required only nicotinic acid; however, A. aceti 295 was able to grow without any added supplements in glucose mineral salts medium. Optimal vitamin concentrations for maximal growth and optimal pH for the maximal number of generations per hour was established for a few pink disease strains.Journal Series Paper No. 2373 of the Hawaii Agricultural Experiment Station, Honolulu.     

Comparison of biological and conventional insecticide treatments for the management of the pineapple fruit borer, Strymon megarus (Lepidoptera: Lycaenidae) in Costa Rica

Carbaryl is currently one of the most commonly used insecticides for the control of the pineapple fruit borer, Strymon megarus (Godart), in commercial pineapple production. To evaluate more sustainable biological alternatives to conventional insecticides, three microbial and one botanical insecticide were studied. Beauveria bassiana, Metarhizium anisopliae, Bacillus thuringiensis (Bt) and a plant extract from Quassia amara were compared with carbaryl in replicated field trials in Costa Rica during 2005 and 2006. In both years of testing, the untreated control received over 50% fruit damage from S. megarus. Bt and carbaryl provided the highest level of control and the lowest level of fruit damage compared to all other treatments. Based on the results of this study, Bt appears to be an acceptable biological alternative to the conventional insecticide (carbaryl) for control of S. megarus on pineapple. In addition, Bt was the least expensive treatment used in this study.     

Isolation and characterization of diazotrophic bacteria from banana and pineapple plants

Banana and pineapple fruit crops are widely cultivated in tropical areas where high amounts of fertilizers are applied, principally nitrogen. Over 200 kg N.ha^-1.yr^-1 is often applied to these crops. Nevertheless, developing countries face the problem of high costs of chemical fertilizers. As already demonstrated for other tropical crops, like sugar cane, the utilization of nitrogen-fixing bacteria may support the growth of these fruit plants. In this work, we demonstrate the association of nitrogen-fixing bacteria with banana and pineapple. Samples from roots, stems, leaves and fruits of different genotypes showed the occurrence of diazotrophic bacteria, when evaluated in semi-specific semi-solid media. These isolates could be separated into seven different groups according to their morphological and physiological characteristics. Additional, phylogenetic assignments were performed with group- and species-specific oligonucleotide probes. Bacteria related to the groups of Azospirillum amazonense, Azospirillum lipoferum, Burkholderia sp. and a group similar to the genus Herbaspirillum could be detected in samples of both crops. However, Azospirillum brasilense and another two groups of Herbaspirillum-like bacteria were detected only in banana plants. Two isolates of the latter group were identified as Herbaspirillum seropedicae, whereas the other isolates may represent a new Herbaspirillum species.     

First morphogenetic identification of <i>Fusarium solani</i> isolated from orange fruit in Egypt

Losses due to postharvest decay may occur at any time during postharvest handling, from harvest to consumption affecting the produce quality and quantity. Accurate identification of the pathogen causing postharvest disease is essential to the selection of an appropriate disease control approach. Nine isolates of Fusarium recovered from orange fruit were identified as Fusarium solani. The fungus is involved with fruit decay. The obtained cultures were purified and grown on potato-dextrose agar (PDA), malt yeast agar (MYA), and Czapek's nutrient media (CNM) under light for identification. A pathogenicity test was carried out to fulfil Koch's postulates. The pathogen could only enter ripe orange fruit through wounds and cracks causing the rot disease. The identification of the fungal isolates was confirmed to be F. solani by DNA sequencing, which was 99 to 100% homologous to those deposited in the Gen- Bank. The identity of nine fungal isolates was confirmed to be F. solani by DNA sequencing of the internal transcribed spacer (ITS) rDNA region (GenBank Accession Nos. DQ486874 to DQ486881 and KC758879). To our knowledge, this is the first morphogenetic identification of F. solani isolated from orange fruit in Egypt.     

Bioassay for in vitro differentiation of pineapple cultivar resistance levels to heart rot disease

Summary An in vitro bioassay to differentiate pineapple plant resistance levels to Phytophthora nicotianae var. parasitica (heart rot disease) is deseribed here. Conditions to cause death of in vitro-cultured plants were defined using a cultivar previously found to be susceptible to this fungus in our Field-Grown Pineapple Germplasm Bank (ev. Smooth Cayenne Serrana). The effects of zoospore concentration, inoculation technique, and disease progress during the course of time after infection were evaluated. The highest rates of plant death were observed with the use of 10⁸ zoospores ml⁻¹, and the inoculation technique of needle-mediated leaf base wound. One hundred percent plant death was observed at 144h after infection. Different susceptible varieties along with a resistant pineapple relative were additionally compared. In vitro results confirmed previous observations obtained under field conditions. The protocol described here may be used for early selection (in vitro) of new pineapple genotypes showing resistance to this fungus. At present, this protocol is extensively used in the Biotechnology-assisted Cuban Program for Pineapple Breeding.     

A procedure for isolating Pyrenochaeta terrestris from onion roots

Surface sterilised stelar tissues from onion roots exhibiting typical pink root symptoms were plated on water agar and incubated under cool white fluorescent light (CWFL) for 12 h day^-1 at 20°C. After 4–5 days, the plates were examined with the 10? objective of a compound microscope and tissues producing conidia of Fusarium spp. were discarded. Sub-cultures of isolates suspected to be Pyrenochaeta terrestris, grown from Fusarium-negative tissues, were transferred to chloroamphenicol-amended (500 ppm) corn-meal (CCMA) agar plates and incubated at 24°C under CWFL for a further 10–14 days. Cultures with setose pycnidia were identified as P. terrestris. Plates were again scanned for conidia of Fusarium spp., and cultures negative for Fusarium spp., but not producing pycnidia, were suspected to be P. terrestris. These were compared with known isolates on CCMA. On this medium growth of P. terrestris was slow, producing appressed, pinkish colonies which were circular with a smooth margin. All isolates of P. terrestris isolated by the procedure caused pink root of onion when tested, whereas none of the Fusarium spp. isolated produced the disease. However, a mixed inoculums of P. terrestris and Fusariurn solani produced typical pink root symptoms.     

Identification and characterization of a Pantoea citrea gene encoding glucose dehydrogenase that is essential for causing pink disease of pineapple.

Pantoea citrea, a member of the family Enterobacteriaceae, causes pink disease of pineapple, whose symptom is characterized by the formation of pink to brown discolorations of the infected portions of the pineapple fruit cylinder upon canning. Molecular genetic approaches were applied to elucidate the mechanism responsible for this fruit discoloration. A P. citrea mutant strain, CMC6, defective in its ability to cause pink disease and fruit discoloration, was generated by nitrosoguanidine mutagenesis. A DNA fragment that restored these activities was isolated by screening a genomic cosmid library of P. citrea. A large open reading frame of 2,361 bp, identified by nucleotide sequencing of a subclone of the complementing DNA, showed high similarities to identified genes encoding glucose dehydrogenase (GDH) in Escherichia coli, Acinetobacter calcoaceticus, and Gluconobacter oxydans. The predicted amino acid sequence of GDH of P. citrea was identical to known GDHs in these bacteria by 54, 44, and 34%, respectively. GDH of P. citrea has a predicted molecular mass of 86.2 kDa, contains a conserved binding domain for the cofactor pyrroloquinoline quinone, and possesses GDH activity as demonstrated by biochemical assay. GDH is the key branch point enzyme leading to the biosynthesis of gluconate, which in turn serves as the substrate leading to the formation of 2-ketogluconate, 2,5-diketogluconate, 6-phosphogluconate, and 2-keto-6-phosphogluconate. Addition of gluconate to CMC6 restores the juice- and fruit-discoloring activity. Although the pigments formed by heating (or canning) have not been identified, it is clear that GDH is one of the enzymes required for pigment formation leading to pink disease.     

地膜/网纱覆盖对冬季菠萝园小环境及菠萝生长和果实品质特性的影响

为探明地膜/网纱覆盖在菠萝冬季防寒中的作用,以露地(NF)栽培为对照,设置地膜+网(FM+GC)、地膜(FM)及露地+网(NF+GC)3种处理,研究了地膜/网纱覆盖对冬季菠萝园小环境温湿度、光照强度、土壤温度的调控效应,及其对冬季菠萝植株、果实生长,果实品质形成等的影响。结果显示,(1)与露地对照相比,地膜+网、地膜及露地+网处理均提高了小环境的空气温度;地膜+网、露地+网均提高了空气湿度,而地膜处理在10:00前可提高空气湿度,11:30后会降低;地膜+网、露地+网处理均降低了光照强度,但地膜处理则可略微提高光照强度;地膜+网、地膜及露地+网处理均提高了土壤温度。(2)地膜+网、地膜及露地+网处理的植株新抽叶片数增多,新抽叶片长度增加,果实单果重及纵、横径提高,但可溶性固形物、可滴定酸含量及固/酸比变化不明显。(3)地膜+网、地膜处理的菠萝果实果皮的L*、a*、b*值提高,而露地+网处理的则降低;地膜+网处理的菠萝果肉L*、a*、b*值提高,而地膜处理的果肉L*值提高,a*、b*值却降低;露地+网处理的菠萝果肉L*、a*、b*值均降低。(4)地膜+网处理的果实酯类香气物质总相对含量降低,烯类总相对含量提高;而地膜、露地+网处理的则相反。研究表明,地膜/网纱覆盖促进了冬季菠萝植株及果实的生长,对果实品质无显著影响,但网纱覆盖对菠萝果实色泽、香气物质有一定的影响。     

Inhibition of penicillium digitatum and penicillium italicum in vitro and in planta with Panomycocin, a novel exo-β-1,3-glucanase isolated from Pichia anomala NCYC 434

Panomycocin, a novel exo-beta 1,3 glucanase, was tested as an antifungal agent against green and blue mold diseases, the most important causes of post harvest decay in citrus fruits. All tested isolates of Penicillium digitatum and Penicillium italicum were susceptible to panomycocin in vitro. Effective panomycocin concentrations for 50% growth inhibition (MIC-2) for P. digitatum and P. italicum were 2 and 1 μg ml⁻¹, respectively. Complete (MIC-0) growth inhibition of all isolates observed at a panomycocin concentration of 16 μg ml⁻¹. Treatment of spores with panomycocin at values lower than the MIC-0 led to slower germ tube elongation and mycelium growth. In tests on fruit, panomycocin at concentrations equal to in vitro MIC-0 value protected lemon fruit from decay.     

Virulence of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae against red palm weevil,Rhynchophorus ferrugineus (Olivier)

Nineteen different isolates of the entomopathogenic fungi Beauveria bassiana sensu lato (s.l.) and Metarhizium anisopliae s.l. (Ascomycota: Hypocreales), recovered from different soil samples (field crops, fruit orchards, vegetable fields and forests) and insect cadavers were tested against Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) at two different spore concentrations (1 × 10⁷ and 1 × 10⁸ conidia mL^?1). Three isolates of B. bassiana and two of M. anisopliae gave >88 % larval and >75 % adult mortality of R. ferrugineus on their highest dose rate respectively. More sporulating cadavers (mycosis) resulted from a high dose rate compared to low dose on both life stages of R. ferrugineus. The current study confirmed the lethal action of B. bassiana and M. anisopliae isolates with mortality levels usually directly proportional to the conidial concentration. This study further confirmed that the isolates recovered from R. ferrugineus dead cadavers gave more mortality compared to the other sources. In the virulence assay two isolates of B. bassiana caused the highest percentage of both larval and adult mortality at all exposure intervals which suggest that they may be the most promising for use in sustainable management programs aimed at microbial control in date palm orchards.     

Pathogenicity of fungi associated with melon vine decline and selection strategies for breeding resistant cultivars

Melone Vine Decline is a severe rot root disease of increasing world‐wide importance. In Eastern Spain it is related to the presence of Acremonium cucurbitacearum and Monosporascus cannonballus. The strong influence of environmental conditions on the progress of this disease has made its study difficult. A field screening of Cucumis melo accessions has been conducted over four years. Simultaneously, the pathogenicity of isolates of the two fungi recovered from the screening field was studied. These were more aggressive than other Spanish and American isolates. Percentage of vine decay was scored, togeter with root damage, the latter being evaluated by using four scoring systems based on root characteristics and disease severity. Root inspection alowed the selection of resistance sources, even when aboveground symptoms did not appear, due to the lack of environmental stresses at time of fruit maturity. The root damage scoring if environmental stresses occur during fruit maturity. The accession C. melo var. agrestis Pat 81 consistently exhibited high field resistance level, expressed as a higher percentage of symptomeless plants, together with a significant delay in symptoms appearance. The F₁ hybrids derived from the cross Pat 81 ×C. melo susceptible varienties showed an intermediate level of resistance between the parents, suggesting a partial dominance gene action. The high resistance level found in Pat 81, and also in its derived hybrids, against the aggressive isolates found in this area, makes it promissing for breeding melon verieties resistant to melon vine decline.     

Effective and Low-Cost Saccharification of Pineapple Peel by <Emphasis Type="Italic">Trichoderma viride</Emphasis> Crude Extract with Enhanced β-Glucosidase Activity

Efficient, low-cost enzymatic hydrolysis of lignocellulosic biomass is essential for cost-effective production of bioethanol. The aim of this study was to establish a fungal fermentation-based strategy for the economic enzymatic conversion of pineapple peel into fermentable sugars. Trichoderma viride was grown on passion fruit peel in order to improve its β-glucosidase production, and a crude extract was then used to hydrolyze pineapple peel. The effects of medium pH, cultivation time, and passion fruit peel concentration on β-glucosidase production were evaluated using a central composite rotational design (CCRD) combined with response surface methodology (RSM). Optimal β-glucosidase activity of 2.40 U mL^?1 was found after 6.5 days of cultivation in medium at pH 6.0, containing 2.0 % passion fruit peel. Saccharification of pineapple peel was also optimized by RSM and CCRD with respect to pH, temperature, β-glucosidase concentration, and reaction time and proceeded optimally at pH 4.0, 55 °C, with a β-glucosidase loading of 31.25 U g^?1 dry feedstock and 75 h of reaction. Under these conditions, T. viride crude extract hydrolyzed pineapple peel with a glucose yield of 65.3 %. This study therefore presents passion fruit peel as an attractive raw material for the production of β-glucosidases. In addition, it describes an improved, effective, and low-cost enzymatic method for the production of fermentable sugars from pineapple peel, an abundant and inexpensive agro-industrial waste.     

Aureobasidium pullulans as a biocontrol agent of postharvest pathogens of apples in Uruguay

Aureobasidium pullulans was the microorganism most frequently recovered from the surface of apple fruit (cv. Red Delicious) stored in commercial cold chambers for 6 months. In the present work, 10 isolates of Aureobasidium pullulans were assayed to determine if they could control blue and grey mold disease of apple during cold storage. Although nine of 10 isolates, significantly reduced the percentage of decayed wounds when compared to the control, one of them, designated isolate ApB, showed the highest levels of protection. ApB was able to grow in a wide range of temperatures lower than 35°C, which is an important human health safety factor. ApB was resistant to thiabendazole, iprodione and imazalil, the most commonly commercially applied fungicides in postharvest treatment of apples in Uruguay. Regarding the mechanisms of action of the selected biocontrol agent, lytic enzymes did not seem to play a central role. ApB depleted iron from nutrient media, which may be an important aspect of its ability to inhibit Botrytis cinerea. Further experiments are needed, however, to determine if the depletion of iron is caused by the production of siderophores, by the immobilization of iron in an insoluble pigment, or a combination of both.     

Control of green mould of citrus by using Trichoderma harzianum,humic acid or garlic

Penicillium digitatum, an aggressive fungus causes post-harvest decay of mandarin sweet orange and Washington navel. In vitro Trichoderma harzianum or humic acid (HA) or powdered cloves of garlic caused inhibition of fungal growth of isolates P₁ and P₂. Under storage conditions, the fruit citrus is protected by using T. harzianum with standard volume 2.0?ml (9.6?×?10⁶?conidia/ml) and application 24?h before inoculation reduces disease incidence and disease severity after seven?days from inoculation with P. digitatum spore suspension (1.0?×?10⁶?spores/ml) compared to control. Spraying the fruit citrus by standard volume of 2.0?ml of either HA or powder cloves of garlic 1% on each fruit 24?h before inoculation reduces disease incidence and disease severity after seven?days from inoculation with P. digitatum (1.0?×?10⁶ spores/ml) compared to control. The lowest percentage of disease incidence and disease severity were associated with powder of cloves garlic and followed by HA and T. harzianum during two growing seasons compared with the untreated and control.