Journal of Microbiology

Journal Article

Nano-encapsulation of naringinase produced by Trichoderma longibrachiatum ATCC18648 on thermally stable biopolymers for citrus juice debittering: Manal M. Housseiny , Heba I. Aboelmagd; J. Microbiol. 2019;57(6):521-531. Published online May 27, 2019; DOI: https://doi.org/10.1007/s12275-019-8528-6

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18 Citations

Abstract: Characteristics of naringinase nano-encapsulated forms on different carrier materials (chitosan and alginate polymers) were investigated in this study. Screening of twelve fungal isolates for naringinase production indicated that Trichoderma longibrachiatum was the most promising. Grapefruit rind was used as a substrate containing naringin for naringinase production. TEM micrographs showed that chitosan nano-capsules were applied for the production of morphologically homogeneous enzymatic nano-particles with high enzyme encapsulation efficiency, small asymmetric sizes (from 15.09 to 27.07 nm with the mean of 21.8 nm) and rough surfaces compared to nano-encapsulated naringinase in alginate which showed nano-particle size (from 33.37 to 51.01 nm with the mean of 43.03 nm). It was revealed that the highest naringinase activity was found in case of chitosan nano-capsule naringinase compared to alginate nano-capsule one. Thermogram analysis (TGA) showed that the free enzyme loses about 92% of its weight at approximately 110°C, while the nanoencapsulated ones show more stability at higher temperatures. Conclusively, the nano-capsulation process improves the kinetics and operational stability so could be useful as a debittering agent for various thermal processing applications in citrus juices industries which makes the fruit juice more acceptable and cost-effective to the consumer.

Research Support, Non-U.S. Gov'ts

Effect of Long-Term Different Fertilization on Bacterial Community Structures and Diversity in Citrus Orchard Soil of Volcanic Ash: Jae Ho Joa , Hang Yeon Weon , Hae Nam Hyun , Young Chull Jeun , Sang Wook Koh; J. Microbiol. 2014;52(12):995-1001. Published online November 29, 2014; DOI: https://doi.org/10.1007/s12275-014-4129-6

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32 Citations

Abstract: This study was conducted to assess bacterial species richness, diversity and community distribution according to different fertilization regimes for 16 years in citrus orchard soil of volcanic ash. Soil samples were collected and analyzed from Compost (cattle manure, 2,000 kg/10a), 1/2 NPK+compost (14-20-14+2,000 kg/10a), NPK+compost (28-40-28+2,000 kg/10a), NPK (28-40-28 kg/10a), 3 NPK (84-120-84 kg/10a), and Control (no fertilization) plot which have been managed in the same manners with compost and different amount of chemical fertilization. The range of pyrosequencing reads and OTUs were 4,687–7,330 and 1,790–3,695, respectively. Species richness estimates such as Ace, Chao1, and Shannon index were higher in 1/2 NPK+compost than other treatments, which were 15,202, 9,112, 7.7, respectively. Dominant bacterial groups at level of phylum were Proteobacteria, Acidobacteria, and Actinobacteria. Those were occupied at 70.9% in 1/2 NPK+compost. Dominant bacterial groups at level of genus were Pseudolabrys, Bradyrhizobium, and Acidobacteria. Those were distributed at 14.4% of a total of bacteria in Compost. Soil pH displayed significantly closely related to bacterial species richness estimates such as Ace, Chao1 (p<0.05) and Shannon index (p<0.01). However, it showed the negative correlation with exchangeable aluminum contents (p<0.05). In conclusion, diversity of bacterial community in citrus orchard soil was affected by fertilization management, soil pH changes and characteristics of volcanic ash.

The Role as Inoculum Sources of Xanthomonas citri pv. citri Surviving on the Infected Satsuma mandarin Fruits: So Young Kang , Ki Deok Kim , Jeum Kyu Hong , He Nam Hyun , Yong Chull Jeun; J. Microbiol. 2014;52(5):422-426. Published online April 11, 2014; DOI: https://doi.org/10.1007/s12275-014-3366-z

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Abstract: Importing citrus fruits infected by Asiatic citrus canker caused by Xanthomonas citri pv. citri (Xcc) can act as an inoculum source for the disease epidemic in citrus canker-free countries. In this study, the pathogenicity of the causal agent of Asiatic citrus canker surviving on infected Satsuma mandarin fruits was evaluated. The washing solution of infected Satsuma mandarin fruits did not cause lesion formation on the citrus leaves. However, a typical citrus canker lesion was formed on the leaves after inoculation with higher concentrations of the inoculum from the washing solution (washing solu-tion II). It indicated that the pathogenicity of the citrus can-ker surviving on the symptomatic Satsuma mandarin fruits was not changed. Scanning electron microscopic observation showed that the numbers of bacterial cells on the leaves of Satsuma mandarin which inoculated with the washing solu-tion directly (washing solution I) was less compared to those of leaves inoculated with the washing solution II. This result supports that the pathogenicity of Xcc surviving on Satsuma mandarin fruits may not be changed but that the sucessful infection of citrus caker may depend on the concentration of the inoculum.

Transmission of Methylobacterium mesophilicum by Bucephalogonia xanthophis for Paratransgenic Control Strategy of Citrus Variegated Chlorosis: Cláudia Santos Gai , Paulo Teixeira Lacava , Maria Carolina Quecine , Marie-Christine Auriac , João Roberto Spotti Lopes , Welington Luiz Araújo , Thomas Albert Miller , João Lúcio Azevedo; J. Microbiol. 2009;47(4):448-454. Published online September 9, 2009; DOI: https://doi.org/10.1007/s12275-008-0303-z

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42 Citations

Abstract: Methylobacterium mesophilicum, originally isolated as an endophytic bacterium from citrus plants, was genetically transformed to express green fluorescent protein (GFP). The GFP-labeled strain of M. mesophilicum was inoculated into Catharanthus roseus (model plant) seedlings and further observed colonizing its xylem vessels. The transmission of this endophyte by Bucephalogonia xanthophis, one of the insect vectors that transmit Xylella fastidiosa subsp. pauca, was verified by insects feeding from fluids containing the GFP bacterium followed by transmission to plants and isolating the endophyte from C. roseus plants. Forty-five days after inoculation, the plants exhibited endophytic colonization by M. mesophilicum, confirming this bacterium as a nonpathogenic, xylem-associated endophyte. Our data demonstrate that M. mesophilicum not only occupy the same niche of X. fastidiosa subsp. pauca inside plants but also may be transmitted by B. xanthophis. The transmission, colonization, and genetic manipulation of M. mesophilicum is a prerequisite to examining the potential use of symbiotic control to interrupt the transmission of X. fastidiosa subsp. pauca, the bacterial pathogen causing Citrus variegated chlorosis by insect vectors.

Research Support, U.S. Gov't, Non-P.H.S.

Isolation and Molecular Characterization of Xylella fastidiosa from Coffee Plants in Costa Rica: Mauricio Montero-Astua , Carlos Chacon-Diaz , Estela Aguilar , Carlos Mario Rodriguez , Laura Garita , William Villalobos , Lisela Moreira , John S. Hartung , Carmen Rivera; J. Microbiol. 2008;46(5):482-490. Published online October 31, 2008; DOI: https://doi.org/10.1007/s12275-008-0072-8

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30 Citations

Abstract: Coffee plants exhibiting a range of symptoms including mild to severe curling of leaf margins, chlorosis and deformation of leaves, stunting of plants, shortening of internodes, and dieback of branches have been reported since 1995 in several regions of Costa Rica''s Central Valley. The symptoms are referred to by coffee producers in Costa Rica as ''crespera'' disease and have been associated with the presence of the bacterium Xylella fastidiosa. Coffee plants determined to be infected by the bacterium by enzyme linked immunosorbent assay (ELISA), were used for both transmission electron microscopy (TEM) and for isolation of the bacterium in PW broth or agar. Petioles examined by TEM contained rod-shaped bacteria inside the xylem vessels. The bacteria measured 0.3 to 0.5 um in width and 1.5 to 3.0 um in length, and had rippled cell walls 10 to 40 nm in thickness, typical of X. fastidiosa. Small, circular, dome-shaped colonies were observed 7 to 26 days after plating of plant extracts on PW agar. The colonies were comprised of Gram-negative rods of variable length and a characteristic slight longitudinal bending. TEM of the isolated bacteria showed characteristic rippled cell walls, similar to those observed in plant tissue. ELISA and PCR with specific primer pairs 272-1-int/272-2-int and RST31/RST33 confirmed the identity of the isolated bacteria as X. fastidiosa. RFLP analysis of the amplification products revealed diversity within X. fastidiosa strains from Costa Rica and suggest closer genetic proximity to strains from the United States of America than to other coffee or citrus strains from Brazil.

Research Support, Non-U.S. Gov'ts

The Endophyte Curtobacterium flaccumfaciens Reduces Symptoms Caused by Xylella fastidiosa in Catharanthus roseus: Paulo Teixeira Lacava , Wenbin Li , Welington Luiz Araujo , Joao Lucio Azevedo , John Stephen Hartung; J. Microbiol. 2007;45(5):388-393.; DOI: https://doi.org/2599 [pii]

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Abstract: Citrus variegated chlorosis (CVC) is a disease of the sweet orange [Citrus sinensis (L.)], which is caused by Xylella fastidiosa subsp. pauca, a phytopathogenic bacterium that has been shown to infect all sweet orange cultivars. Sweet orange trees have been occasionally observed to be infected by Xylella fastidiosa without evidencing severe disease symptoms, whereas other trees in the same grove may exhibit severe disease symptoms. The principal endophytic bacterial species isolated from such CVC-asymptomatic citrus plants is Curtobacterium flaccumfaciens. The Madagascar periwinkle [Citrus sinensis (L.)] is a model plant which has been used to study X. fastidiosa in greenhouse environments. In order to characterize the interactions of X. fastidiosa and C. flaccumfaciens, periwinkle plants were inoculated separately with C. flaccumfaciens, X. fastidiosa, and both bacteria together. The number of flowers produced by the plants, the heights of the plants, and the exhibited disease symptoms were evaluated. PCR-primers for C. flaccumfaciens were designed in order to verify the presence of this endophytic bacterium in plant tissue, and to complement an existing assay for X. fastidiosa. These primers were capable of detecting C. flaccumfaciens in the periwinkle in the presence of X. fastidiosa. X. fastidiosa induced stunting and reduced the number of flowers produced by the periwinkle. When C. flaccumfaciens was inoculated together with X. fastidiosa, no stunting was observed. The number of flowers produced by our doubly- inoculated plants was an intermediate between the number produced by the plants inoculated with either of the bacteria separately. Our data indicate that C. flaccumfaciens interacted with X. fastidiosa in C. roseus, and reduced the severity of the disease symptoms induced by X. fastidiosa. Periwinkle is considered to be an excellent experimental system by which the interaction of C. flaccumfaciens and other endophytic bacteria with X. fastidiosa can be studied.

Evaluation of Endophytic Colonization of Citrus sinensis and Catharanthus roseus Seedlings by Endophytic Bacteria: Paulo Teixeira Lacava , Welington Luiz Araujo , Joao Lucio Azevedo; J. Microbiol. 2007;45(1):11-14.; DOI: https://doi.org/2498 [pii]

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Abstract: Over the last few years, the endophytic bacterial community associated with citrus has been studied as an important component interacting with Xylella fastidiosa, the causal agent of citrus variegated chlorosis (CVC). This bacterium may also colonize some model plants, such as Catharanthus roseus and Nicotiana clevelandii. In the present study, we compared the endophytic colonization of Citrus sinensis and Catharanthus roseus using the endophytic bacteria Klebsiella pneumoniae. We chose an appropriate strain, K. pneumoniae 342 (Kp342), labeled with the GFP gene. This strain was inoculated onto seedlings of C. sinensis and C. roseus. The isolation frequency was determined one week after the inoculation and the endophytic colonization of K. pneumoniae was observed using fluorescence microscopy. Although the endophytic bacterium was more frequently isolated from C. roseus than from C. sinensis, the colonization profiles for both host plants were similar, suggesting that C. roseus could be used as a model plant to study the interaction between endophytic bacteria and X. fastidiosa.

Impact of Genetically Modified Enterobacter cloacae on Indigenous Endophytic Community of Citrus sinensis Seedlings: Fernando Dini Andreote , Marcelo Jose Mortatti Gullo , Andre Oliveira de Souza Lima , Walter Maccheroni Junior , Joao Lucio Azevedo , Welington Luiz Araujo; J. Microbiol. 2004;42(3):169-173.

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Abstract: Enterobacter cloacae (strain PR2/7), a genetically modified endophyte (GME) in citrus plants, carrying different plasmids (pEC3.0/18, pCelE, pEglA and pGFP), was inoculated into Citrus sinensis seedlings under greenhouse conditions. The impact of this on the indigenous bacterial endophytic community was studied by analyses of 2 different morphologic groups. The germination rates of inoculated seeds were evaluated in greenhouse, and plasmid stability under in vitro conditions. Results demonstrated a great and diverse endophytic community inside plants, and specialization in tissue colonization by some bacterial groups, in different treatments. Shifts in seed germination rate were observed among treatments: in general, the PR2/7 harboring pEglA bacterial clone significantly reduced seed germination, compared to the PR2/7 harboring pEC3.0/18 clone. This suggests that the presence of the pEglA plasmid changes bacteria-seed interactions. The endophytic community of citrus seedlings changed according to treatment. In seedlings treated with the PR2/7 with pEglA clone, the population of group II decreased significantly, within the context of the total endophytic community. These results indicate that the application of GMEs induces shifts in the endophytic bacterial community of citrus seedlings.

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