Diversity of endophytic bacterial populations and their interaction with Xylella fastidiosa in citrus plants

Citrus variegated chlorosis (CVC) is caused by Xylella fastidiosa, a phytopathogenic bacterium that can infect all Citrus sinensis cultivars. The endophytic bacterial communities of healthy, resistant, and CVC-affected citrus plants were studied by using cultivation as well as cultivation-independent techniques. The endophytic communities were assessed in surface-disinfected citrus branches by plating and denaturing gradient gel electrophoresis (DGGE). Dominant isolates were characterized by fatty-acid methyl ester analysis as Bacillus pumilus, Curtobacterium flaccumfaciens, Enterobacter cloacae, Methylobacterium spp. (including Methylobacterium extorquens, M. fujisawaense, M. mesophilicum, M. radiotolerans, and M. zatmanii), Nocardia sp., Pantoea agglomerans, and Xanthomonas campestris. We observed a relationship between CVC symptoms and the frequency of isolation of species of Methylobacterium, the genus that we most frequently isolated from symptomatic plants. In contrast, we isolated C. flaccumfaciens significantly more frequently from asymptomatic plants than from those with symptoms of CVC while P. agglomerans was frequently isolated from tangerine (Citrus reticulata) and sweet-orange (C. sinensis) plants, irrespective of whether the plants were symptomatic or asymptomatic or showed symptoms of CVC. DGGE analysis of 16S rRNA gene fragments amplified from total plant DNA resulted in several bands that matched those from the bacterial isolates, indicating that DGGE profiles can be used to detect some endophytic bacteria of citrus plants. However, some bands had no match with any isolate, suggesting the occurrence of other, nonculturable or as yet uncultured, endophytic bacteria. A specific band with a high G+C ratio was observed only in asymptomatic plants. The higher frequency of C. flaccumfaciens in asymptomatic plants suggests a role for this organism in the resistance of plants to CVC.     

Influence of Xylella fastidiosa infection of citrus on host selection by leafhopper vectors

Infection of plants by pathogens can influence their attractiveness and suitability to insect vectors and other herbivores. Here we examined the effects of Citrus sinensis (L.) Osbeck (Rutaceae) infection by the bacterium Xylella fastidiosa, which causes citrus variegated chlorosis (CVC), on the feeding preferences of two sharpshooter vectors, Dilobopterus costalimai Young and Oncometopia facialis (Signoret) (Homoptera: Cicadellidae). Experiments were performed inside observation chambers, in which a healthy plant and an infected one (with or without CVC symptoms) were supplied to a group of 40 sharpshooters. The number of insects that selected each treatment was recorded at several time intervals in 48 h. In another experiment, the ingestion rate on healthy and infected (symptomatic or not) plants was evaluated by measuring the liquid excretion of sharpshooters that were confined on branches of each plant for 72 h. Both sharpshooter species preferred healthy plants to those with CVC symptoms. However, O. facialis did not discriminate between healthy citrus and symptomless infected plants. Feeding by D. costalimai was markedly reduced when confined on CVC‐symptomatic plants, but not on asymptomatic infected ones. The ingestion rate by O. facialis was not affected by the presence of CVC symptoms. The results suggest that citrus trees with early (asymptomatic) infections by X. fastidiosa may be more effective as inoculum sources for CVC spread by insect vectors than those with advanced symptoms.     

Biological Control of Bacterial Wilt of Bean Using a Bacterial Endophyte, Pantoea agglomerans

Indoor studies were conducted to determine the potential use of Pantoea agglomerans isolate LRC 8311 as a biocontrol agent for control of bacterial wilt of bean caused by Curtobacterium flaccumfaciens pv. flaccumfaciens. Soaking seeds of great northern bean cv. US1140 in a suspension of 3 × 10⁸ cfu/ml P. agglomerans resulted in thorough endophytic colonization of the entire bean seedling from root to apical stem after 7 days, regardless of whether the inoculated seeds were hilum injured or not. Colonization of seedlings by P. agglomerans increased seedling height after 10 days, and had no negative effect on seedling emergence. Treatment of hilum‐injured bean seeds of great northern bean cv. US1140 or navy bean cv. Morden003 with a mixture of P. agglomerans + C. flaccumfaciens pv. flaccumfaciens resulted in a high rate of colonization of seedlings by P. agglomerans, reduced frequency of infection by C. flaccumfaciens pv. flaccumfaciens, improved seedling emergence and height, and reduced disease severity, compared with seeds treated with the wilt pathogen alone. Application of P. agglomerans as a soil drench 24 h after planting was also effective in suppressing bacterial wilt in some instances, but was generally not as effective as seed treatment. The study suggests that seed treatment with P. agglomerans may be an effective and practical method for control of bacterial wilt of bean.     

Analysis of the bacterial community in glassy-winged sharpshooter heads

The glassy‐winged sharpshooter (GWSS), Homalodisca vitripennis, is an important vector of various strains of Xylella fastidiosa, which cause disease in a variety of economically important plants. These diseases include citrus variegated chlorosis, oleander leaf scorch and Pierce's Disease of grapevines. Symbiotic control (SC) is a new strategy that uses symbiotic endophytes as biological control agents to antagonize or displace the pathogenic strains of X. fastidiosa. Candidate endophytes for use in SC must occupy the xylem of host plants and attach to the pre‐cibarium and cibarium of sharpshooter insects in order to have access to the pathogen. The study of the bacterial community of GWSS heads by isolation and denaturing gradient gel electrophoresis (DGGE) revealed the presence of species that may be suitable for use in SC. In addition, the results indicated that two important factors, insect age and choice of host plant, affect the composition of the bacterial community in GWSS heads. The main bacterial genera isolated as colonizers of GWSS heads were identified, using partial 16S rRNA gene sequencing, as Bacillus, Pseudomonas, Pedobacter and Methylobacterium, as well as the species Curtobacterium flaccumfaciens. DGGE patterns revealed a diversity of endophytic species able to colonize the GWSS head. The main genera isolated in culture were also identified using this technique. Principal component analysis (PCA) from polymerase chain reaction (PCR)‐DGGE patterns indicated that the bacteria inhabiting the GWSS head are similar to those found as endophytes inside the host plants, and that insect developmental stage and preferential feeding on one host plant species over another are important factors in determining the composition of the bacterial community in the GWSS head. However, a shift in host plants for a small period of time did not cause changes in the compositions of these communities.     

Evaluation of endophytic colonization of Citrus sinensis and Catharanthus roseus seedlings by endophytic bacteria

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.     

Colonization of Madagascar periwinkle (Catharanthus roseus), by endophytes encoding gfp marker

This study reports the introduction of gfp marker in two endophytic bacterial strains (Pantoea agglomerans C33.1, isolated from cocoa, and Enterobacter cloacae PR2/7, isolated from citrus) to monitor the colonization in Madagascar perinwinkle (Catharanthus roseus). Stability of the plasmid encoding gfp was confirmed in vitro for at least 72 h of bacterial growth and after the colonization of tissues, under non-selective conditions. The colonization was observed using fluorescence microscopy and enumeration of culturable endophytes in inoculated perinwinkle plants that grew for 10 and 20 days. Gfp-expressing strains were re-isolated from the inner tissues of surface-sterilized roots and stems of inoculated plants, and the survival of the P. agglomerans C33:1gfp in plants 20 days after inoculation, even in the absence of selective pressure, suggests that is good colonizer. These results indicated that both gfp-tagged strains, especially P. agglomerans C33.1, may be useful tools to deliver enzymes or other proteins in plant.     

Interaction between endophytic bacteria from citrus plants and the phytopathogenic bacteria Xylella fastidiosa, causal agent of citrus-variegated chlorosis

AIMS: To isolate endophytic bacteria and Xylella fastidiosa and also to evaluate whether the bacterial endophyte community contributes to citrus-variegated chlorosis (CVC) status in sweet orange (Citrus sinensis [L.] Osbeck cv. Pera). METHODS AND RESULTS: The presence of Xylella fastidiosa and the population diversity of culturable endophytic bacteria in the leaves and branches of healthy, CVC-asymptomatic and CVC-symptomatic sweet orange plants and in tangerine (Citrus reticulata cv. Blanco) plants were assessed, and the in vitro interaction between endophytic bacteria and X. fastidiosa was investigated. There were significant differences in endophyte incidence between leaves and branches, and among healthy, CVC-asymptomatic and CVC-symptomatic plants. Bacteria identified as belonging to the genus Methylobacterium were isolated only from branches, mainly from those sampled from healthy and diseased plants, from which were also isolated X. fastidiosa. CONCLUSIONS: The in vitro interaction experiments indicated that the growth of X. fastidiosa was stimulated by endophytic Methylobacterium extorquens and inhibited by endophytic Curtobacterium flaccumfaciens. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides the first evidence of an interaction between citrus endophytic bacteria and X. fastidiosa and suggests a promising approach that can be used to better understand CVC disease.     

The endophyte Curtobacterium flaccumfaciens reduces symptoms caused by Xylella fastidiosa in Catharanthus roseus

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.     

Transmission of <Emphasis Type="Italic">Methylobacterium mesophilicum</Emphasis> by <Emphasis Type="Italic">Bucephalogonia xanthophis</Emphasis> for paratransgenic control strategy of Citrus variegated chlorosis

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.     

Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases

A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension. The gene, designated csa, was isolated from a cDNA expression library. The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts. The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals.     

Variation in Radopholus citrophilus Population Densities in the Citrus Rootstock Carrizo Citrange

Seedlings of the hybrid citrus rootstock, Carrizo citrange (Citrus sinensis × Poncirus trifoliata) do not uniformly limit development of the citrus burrowing nematode, Radopholus citrophilus. Variation in nematode population densities in roots of seedlings germinating from the same seed suggests that factors responsible for nematode incompatibility are not functional or are not inherited uniformly among progeny. Seeds which produced a single seedling were more likely to produce plants which suppressed citrus burrowing nematode population increase than were seeds which produced two or three seedlings.     

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Methylobacterium-Induced Endophyte Community Changes Correspond with Protection of Plants against Pathogen Attack

Plant inoculation with endophytic bacteria that normally live inside the plant without harming the host is a highly promising approach for biological disease control. The mechanism of resistance induction by beneficial bacteria is poorly understood, because pathways are only partly known and systemic responses are typically not seen. The innate endophytic community structures change in response to external factors such as inoculation, and bacterial endophytes can exhibit direct or indirect antagonism towards pathogens. Earlier we showed that resistance induction by an endophytic Methylobacterium sp. in potato towards Pectobacterium atrosepticum was dependent on the density of the inoculum, whereas the bacterium itself had no antagonistic activity. To elucidate the role of innate endophyte communities in plant responses, we studied community changes in both in vitro and greenhouse experiments using various combinations of plants, endophyte inoculants, and pathogens. Induction of resistance was studied in several potato (Solanum tuberosum L.) cultivars by Methylobacterium sp. IMBG290 against the pathogens P. atrosepticum, Phytophthora infestans and Pseudomonas syringae pv. tomato DC3000, and in pine (Pinus sylvestris L.) by M. extorquens DSM13060 against Gremmeniella abietina. The capacities of the inoculated endophytic Methylobacterium spp. strains to induce resistance were dependent on the plant cultivar, pathogen, and on the density of Methylobacterium spp. inoculum. Composition of the endophyte community changed in response to inoculation in shoot tissues and correlated with resistance or susceptibility to the disease. Our results demonstrate that endophytic Methylobacterium spp. strains have varying effects on plant disease resistance, which can be modulated through the endophyte community of the host.     

A Suitable <Emphasis Type="Italic">Xylella fastidiosa</Emphasis> CVC Strain for Post-Genome Studies

The genome sequence of the pathogen Xylella fastidiosa Citrus Variegated Chlorosis (CVC) strain 9a5c has revealed many genes related to pathogenicity mechanisms and virulence determinants. However, strain 9a5c is resistant to genetic transformation, impairing mutant production for the analysis of pathogenicity mechanisms and virulence determinants of this fastidious phytopathogen. By screening different strains, we found out that cloned strains J1a12, B111, and S11400, all isolated from citrus trees affected by CVC, are amenable to transformation, and J1a12 has been used as a model strain in a functional genomics program supported by FAPESP (São Paulo State Research Foundation). However, we have found that strain J1a12, unlike strains 9a5c and B111, was incapable of inducing CVC symptoms when inoculated in citrus plants. We have now determined that strain B111 is an appropriate candidate for post-genome studies of the CVC strain of X. fastidiosa.     

An Effective and Low-Cost Culture Medium for Isolation and Growth of Xylella fastidiosa from Citrus and Coffee Plants

Buffered charcoal–yeast extract medium (BCYE) has been used for isolation of Xylella fastidiosa from citrus (Citrus sinensis) and coffee (Coffea arabica) plants affected by citrus variegated chlorosis (CVC) and coffee leaf scorch (CLS). BCYE is composed of ACES (2-[2-amino-2oxoethyl) amino]-ethanesulfonic acid) buffer, activated charcoal, yeast extract, L-cysteine, ferric pyrophosphate, and agar. ACES buffer is costly and not always commercially available in Brazil, and the L-cysteine and ferric pyrophosphate need to be filter sterilized in 0.22-μm pore membranes before inclusion in the medium. Omission of L-cysteine, addition of magnesium sulfate, and replacements of ACES and ferric pyrophosphate for potassium phosphate and ferrous sulfate resulted in an effective, less expensive, and entirely autoclavable medium, named phosphate buffered charcoal-yeast extract medium (PCYE). The final cost of PCYE was approximately one tenth that of BCYE. Its effectiveness was tested for the isolation of X. fastidiosa from symptomatic leaves collected from 52 citrus plants affected by CVC and 43 coffee plants affected by CLS. PCYE was as effective as BCYE and has been used routinely in our and other laboratories for isolation, growth, and quantification of X. fastidiosa from plant tissues.     

Proteomic changes of Citrus roots in response to long-term manganese toxicity

Key message

Fifty-three and thirty-nine differentially expressed protein spots were isolated from Mn-toxic Citrus sinensis and Citrus grandis roots, respectively. Mn-toxicity-induced changes in protein profiles greatly differed between the two species.

Abstract

Limited information is available on the manganese (Mn)-toxicity-responsive proteins in plant roots. ‘Sour pummelo’ (Citrus grandis) and ‘Xuegan’ (Citrus sinensis) seedlings were irrigated for 17 weeks with 2 (control) or 600 μM (Mn-toxic) MnSO₄. C. sinensis displayed more tolerance to Mn-toxicity than C. grandis, which may be related to more Mn accumulation in roots and less Mn distribution in shoots. Using two-dimensional electrophoresis (2-DE), we isolated 11 up-regulated and 42 down-regulated protein spots from Mn-toxic C. sinensis roots, and 25 up-regulated and 14 down-regulated protein spots from Mn-toxic C. grandis roots. This indicated more metabolic flexibility in C. sinensis roots, thus contributing to the Mn-tolerance of C. sinensis. According to the biological functional properties, these differentially expressed proteins in the two species were classified into the following categories: protein metabolism, nucleic acid metabolism, carbohydrate and energy metabolism, stress responses, cell wall and cytoskeleton, cell transport, signal transduction and fatty acid metabolism. Under Mn-toxicity, proteins involved in nucleic acid metabolism, glycolysis and cell transport were up-regulated in nontolerant C. grandis roots, and down-regulated in tolerant C. sinensis roots. The notable down-regulation of proteins in Mn-toxic C. sinensis roots with less accumulation of carbohydrates may provide an advantage to the net carbon balance by lowering related metabolic processes, and enhancing the Mn-tolerance of C. sinensis. To conclude, there are many important differences in Mn-toxicity-induced changes in protein profiles and metabolic responses between the two species.     

柑橘内生真菌的分离鉴定及其发酵产物对柑橘溃疡病菌的抑制活性

本研究从柑橘抗病品种的健康植株不同组织中分离纯化和鉴定内生真菌,并测定其发酵产物对柑橘溃疡病菌的抑制活性,以明确柑橘抗病品种中内生真菌的组成及其产抗柑橘溃疡病菌活性代谢产物的潜力,为柑橘溃疡病抗菌剂的开发奠定基础.该研究通过组织培养法分离内生真菌,采用形态学和分子生物学方法对其进行鉴定;基于前期的拮抗预试验结果,选取代...     

Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination

The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In citrus, selection using the selectable marker gene nptII, that confers resistance to the antibiotic kanamycin, is in general very effective. An attractive alternative is offered by the MAT system (Multi-Auto-Transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with a MAT vector has been attempted in two citrus genotypes, Pineapple sweet orange (Citrus sinensis L. Osb.) and Carrizo citrange (C. sinensis L. Osb. × Poncirus trifoliata L. Raf.). Results indicated that the IPT phenotype was clearly distinguishable in sweet orange but not in citrange, and that excision was not always efficient and precise. Nevertheless, the easy visual detection of the IPT phenotype combined with the higher transformation efficiency achieved in sweet orange using this system open interesting perspectives for the generation of marker-free transgenic citrus plants.