Quantification of Xylella fastidiosa from Citrus Trees by Real-Time Polymerase Chain Reaction Assay

ABSTRACT Xylella fastidiosa is the causal agent of citrus variegated chlorosis (CVC), a destructive disease of sweet orange cultivars in Brazil. Polymerase chain reaction (PCR)-based assays constitute the principal diagnostic method for detection of these bacteria. In this work, we established a real-time quantitative PCR (QPCR) assay to quantify X. fastidiosa in naturally and artificially infected citrus. The X. fastidiosa cell number detected in the leaves increased according to the age of the leaf, and bacteria were not detected in the upper midrib section in young leaves, indicating temporal and spatial distribution patterns of bacteria, respectively. In addition, the X. fastidiosa cell number quantified in leaves of 'Pera' orange and 'Murcott' tangor reflected the susceptible and resistant status of these citrus cultivars. None of the 12 endophytic citrus bacteria or the four strains of X. fastidiosa nonpathogenic to citrus that were tested showed an increase in the fluorescence signal during QPCR. In contrast, all 10 CVC-causing strains exhibited an increase in fluorescence signal, thus indicating the specificity of this QPCR assay. Our QPCR provides a powerful tool for studies of different aspects of the Xylella-citrus interactions, and can be incorporated into breeding programs in order to select CVC-resistant plants more quickly.     

Seed transmission of Sweet potato leaf curl virus in sweet potato (Ipomoea batatas)

Sweet potato leaf curl virus (SPLCV) infects sweet potato and is a member of the family Geminiviridae (genus Begomovirus). SPLCV transmission occurs from plant to plant mostly via vegetative propagation as well as by the insect vector Bemisia tabaci. When sweet potato seeds were planted and cultivated in a whitefly‐free greenhouse, some sweet potato plants started to show SPLCV‐specific symptoms. SPLCV was detected by PCR from all leaves and floral tissues that showed leaf curl disease symptoms. More than 70% of the seeds harvested from SPLCV‐infected sweet potato plants tested positive for SPLCV. SPLCV was also identified from dissected endosperm and embryos. The transmission level of SPLCV from seeds to seedlings was up to 15%. Southern blot hybridization showed SPLCV‐specific single‐ and double‐stranded DNAs in seedlings germinated from SPLCV‐infected seeds. Taken altogether, the results show that SPLCV in plants of the tested sweet potato cultivars can be transmitted via seeds and SPLCV DNA can replicate in developing seedlings. This is the first seed transmission report of SPLCV in sweet potato plants and also, to the authors' knowledge, the first report of seed transmission for any geminivirus.     

Seven years of negative detection results confirm that Xylella fastidiosa,the causal agent of CVC,is not transmitted from seeds to seedlings

Knowledge about the mechanism of transmission of systemic pathogens of citrus species is highly important for the safe movement of citrus germplasm, management of citrus mother trees, and also production of young plants. Among systemic pathogens of citrus, Xylella fastidiosa the causal agent of the citrus variegated chlorosis (CVC), is one the most important pathogens causing decline in tree vigour and productivity. Seven-year experiments were conducted to evaluate the hypothesis of seed-to-seedling transmission of X. fastidiosa. This bacterium was found colonizing the fruit (exocarp, central axis and mesocarp) and the seed parts (seed coat and endosperm plus embryo). After 7 years of PCR assay, no positive PCR detection of X. fastidiosa was confirmed in seedlings propagated from the seeds infected with X. fastidiosa. This result demonstrates the lack of seed-to-seedling transmission of this bacterium.     

Growth- and leaf-temperature effects on photosynthesis of sweet orange seedlings infected with Xylella fastidiosa

The effects of growth and leaf temperature on photosynthesis were evaluated in sweet orange seedlings ( Citrus sinensis cv. Pera) infected with Xylella fastidiosa (the bacterium that causes citrus variegated chlorosis, CVC). Measurements of leaf gas exchange and chlorophyll  a fluorescence were taken at leaf temperatures of 25, 30, 35 and 40°C in healthy and infected (without visible symptoms) seedlings submitted to two temperature regimes (25/20 or 35/20°C, day/night), not simultaneously. The CO₂ assimilation rates ( A ) and stomatal conductance ( g _s) were higher in healthy plants in both temperature regimes. Values for A and g _s of infected and healthy plants were higher in the 35/20°C regime, decreasing with leaf temperature increase. In addition, differences between healthy and infected plants were higher at 35/20°C, while no differences in chlorophyll  a fluorescence parameters were observed except for potential quantum efficiency of photosystem II, which was higher in infected plants. Low A values in infected plants were caused by low g _s and probably by biochemical damage to photosynthesis. The high alternative electron sink of infected plants was another effect of reduced A . Both high growth and high leaf temperatures increased differences in A between healthy and infected plants. Therefore this feature may be partially responsible for lower growth and/or productivity of CVC-affected plants in regions with high air temperature.     

Colonization of citrus seed coats by 'Candidatus Liberibacter asiaticus': implications for seed transmission of the bacterium

Huanglongbing is an economically damaging disease of citrus associated with infection by 'Candidatus Liberibacter asiaticus'. Transmission of the organism via infection of seeds has not been demonstrated but is a concern since some citrus varieties, particularly those used as rootstocks in commercial plantings are propagated from seed. We compared the incidence of detection of 'Ca. Liberibacter asiaticus' DNA in individual fruit peduncles, seed coats, seeds, and in germinated seedlings from 'Sanguenelli' sweet orange and 'Conners' grapefruit fruits sampled from infected trees. Using real-time quantitative PCR (qPCR) we detected pathogen DNA in nucleic acid extracts of 36 and 100% of peduncles from 'Sanguenelli' and from 'Conners' fruits, respectively. We also detected pathogen DNA in extracts of 37 and 98% of seed coats and in 1.6 and 4% of extracts from the corresponding seeds of 'Sanguenelli' and 'Conners', respectively. Small amounts of pathogen DNA were detected in 10% of 'Sanguenelli' seedlings grown in the greenhouse, but in none of 204 extracts from 'Conners' seedlings. Pathogen DNA was detected in 4.9% and in 89% of seed coats peeled from seeds of 'Sanguenelli' and 'Conners' which were germinated on agar, and in 5% of 'Sanguenelli' but in none of 164 'Conners' seedlings which grew from these seeds on agar. No pathogen DNA was detected in 'Ridge Pineapple' tissue at 3 months post-grafting onto 'Sanguenelli' seedlings, even when pathogen DNA had been detected initially in the 'Sanguenelli' seedling. Though the apparent colonization of 'Conners' seeds was more extensive and nearly uniform compared with 'Sanguenelli' seeds, no pathogen DNA was detected in 'Conners' seedlings grown from these seeds. For either variety, no association was established between the presence of pathogen DNA in fruit peduncles and seed coats and in seedlings.     

Causal Role of Xylella fastidiosa in Oleander Leaf Scorch Disease

ABSTRACT A lethal leaf scorch disease of oleander (Nerium oleander) appeared in southern California in 1993. A bacterium, Xylella fastidiosa, was detected by culturing, enzyme-linked immunoassay, and polymerase chain reaction in most symptomatic plants but not in symptomless plants or negative controls. Inoculating oleanders mechanically with X. fastidiosa cultures from diseased oleanders caused oleander leaf scorch (OLS) disease. The bacterium was reisolated from inoculated plants that became diseased. Three species of xylem sap-feeding leafhoppers transmitted the bacterium from oleander to oleander. The bacterium multiplied, moved systemically, and caused wilting in Madagascar periwinkle (Catharanthus rosea) and leaf scorch in periwinkle (Vinca major) in a greenhouse after inoculation with needle puncture. No bacterium was reisolated from grapevine (Vitis vinifera), peach (Prunus persica), olive (Olea europaea), California blackberry (Rubus ursinus), or valley oak (Quercus lobata) mechanically inoculated with OLS strains of X. fastidiosa. A 500-bp sequence of the 16S-23S ribosomal intergenic region of oleander strains showed 99.2% identity with Pierce's disease strains, 98.4% identity with oak leaf scorch strains, and 98.6% identity with phony peach, plum leaf scald, and almond leaf scorch strains.     

Grapevine phenolic compounds in xylem sap and tissues are significantly altered during infection by Xylella fastidiosa

Pierce's disease of grapevine (PD), caused by the bacterial pathogen Xylella fastidiosa, remains a serious problem for grape production in California and elsewhere. This research examined induction of phenolic compounds in grapevines ('Thompson Seedless') infected with X. fastidiosa over a 6-month period. Two months postinoculation with X. fastidiosa, catechin, digalloylquinic acid, and astringin were found at greater levels in xylem sap; multiple catechins, procyanidins, and stilbenoids were found at greater levels in xylem tissues; and precursors to lignin and condensed tannins were found at greater levels in xylem cell walls. However, such large-scale inductions of phenolic compounds were not observed 4 months after inoculation. Six months after inoculation, infected plants had significantly reduced phenolic levels in xylem sap and tissues when compared with control plants, including lowered levels of lignin and condensed tannins. At 6 months, PD symptoms were severe in infected plants and most photosynthetic tissue was abscised. These results suggest that, even though grapevine hosts may initially respond to X. fastidiosa infections with increased production of phenolic compounds, ultimately, PD causes grapevines to enter a state of decline whereby diseased hosts no longer have the resources to support secondary metabolite production, including defense-associated phenolic compounds.     

Analysis of resistance to Xylella fastidiosa within a hybrid population of Pera sweet orange × Murcott tangor

Resistance to Xylella fastidiosa was evaluated within a population of 20 interspecific hybrids of Pera sweet orange and Murcott tangor under greenhouse conditions. Efficiency of inoculation, multiplication of bacteria within the plants, xylem vessel morphology, and symptom expression were analysed. The rate of infection ranged from 40 to 100% (average 70%) for all genotypes analysed. Xylella fastidiosa populations ranged from log 0·59 to log 2·13 cells mg⁻¹ tissue for the resistant hybrids. These values were significantly different ( P  = 0·05) from those obtained for the tolerant (no symptoms but bacteria recovered) or susceptible (symptoms and bacteria recovered) hybrids (log 3·02 to log 4·06 cells mg⁻¹). Xylella fastidiosa was recovered from all hybrids (log 2·31 to 5·03 CFU mg⁻¹ tissue) except the resistant ones. The first foliar symptoms appeared at least 90 days post-inoculation, the time varying according to genotype. No correlation between xylem vessel morphology and disease expression was observed, indicating that the resistance was the result of a genetic response of the host. According to this hypothesis, a high broad-based heritability index for resistance was obtained (0·96) at 210 days from X. fastidiosa inoculations, using bacterial quantification by real-time PCR, which indicated that the influence of the number of bacteria was the result of genetic rather than environmental variations.     

Endophytes of <Emphasis Type="Italic">Lippia citriodora</Emphasis> (Syn. <Emphasis Type="Italic">Aloysia triphylla</Emphasis>) enhance its growth and antioxidant activity

Sweet pepper (Capsicum annuum) is a popular crop worldwide and an asymptomatic host of the begomovirus (Geminiviridae) Tomato yellow leaf curl virus (TYLCV). A previous study showed that TYLCV could be transmitted by the seeds of tomato plants, but this phenomenon has not been confirmed in other plants. In 2015, four different cultivars of sweet pepper (‘Super Yellow,’ ‘Super Red,’ ‘Sunnyez’ and ‘Cupra’) known to be susceptible to TYLCV were agro-inoculated with a TYLCV infectious clone. Three months after inoculation, the leaves of the ‘Super Yellow’ cultivar showed 80% (8/10) susceptibility and the other three sweet pepper cultivars showed 30 to 50% susceptibilities. All of the ‘Super Yellow’ seed bunches (five seeds per bunch) from plants whose leaves were confirmed to be TYLCV-infected were also TYLCV-infected (8/8). The seeds of other cultivars showed 20 to 40% susceptibilities. Virus transmission rates were also verified with 10 bunches of seedlings for each cultivar (five seedlings per pool). Eight bunches of ‘Super Yellow’ seedlings (8/10) were confirmed to be TYLCV-infected and one to three bunches of each of the other cultivar seedlings were also infected. Viral replication in TYLCV-infected seeds and seedlings was confirmed via strand-specific amplification using virion-sense- and complementary-sense-specific primer sets. This is the first report of TYLCV seed transmission in sweet pepper plants and among non-tomato plants. Because sweet pepper is an asymptomatic host of TYLCV, seeds infected with TYLCV could act as a silent invader of tomatoes and other crops.     

Genetic Diversity of Xylella fastidiosa Strains from Costa Rica, São Paulo, Brazil, and United States

ABSTRACT The diversity of 42 Xylella fastidiosa strains from Costa Rica, S?o Paulo, Brazil, and the United States were analyzed using the sequence of the 16S rRNA gene by variable number of tandem repeat (VNTR) fragment analysis and by restriction fragment length polymorphisms (RFLP) of a specific polymerase chain reaction (PCR)-amplification product using enzyme CfoI. Limited variability in the sequence of the 16S rRNA gene was observed and, although the separation was not absolute, most strains from Costa Rica clustered with strains from the United States and not with strains from S?o Paulo. The PCR-RFLP produced different patterns of DNA bands. The same pattern was shared by strains from Costa Rica, the United States, and two coffee strains from S?o Paulo, but a different pattern was observed in six coffee and orange strains from Brazil. In all, 32 amplification products were scored in the VNTR fragment analysis. The total variation observed among the X. fastidiosa strains had significant (P < 0.001) contributions from both geography and host origin as inferred by Nei's values of genetic diversity and WINAMOVA statistics. The strains from Costa Rica were isolated from diseased grapevines, coffee, and sweet orange and these strains grouped together and could be distinguished from strains from grapevine from the United States or from either coffee or sweet orange from S?o Paulo. The strains tested from Costa Rica are most likely of local origin, although the possibility that they have been introduced along with horticultural crops cannot be excluded. In either case, they are examples of independent selection of strains of X. fastidiosa affecting coffee and sweet orange. Greater genetic similarity was observed between strains from Costa Rica and the United States than with those from S?o Paulo.     

An Evaluation of the Genetic Diversity of Xylella fastidiosa Isolated from Diseased Citrus and Coffee in São Paulo, Brazil

ABSTRACT Strains of Xylella fastidiosa, isolated from sweet orange trees (Citrus sinensis) and coffee trees (Coffea arabica) with symptoms of citrus variegated chlorosis and Requeima do Café, respectively, were indistinguishable based on repetitive extragenic palindromic polymerase chain reaction (PCR) and enterobacterial repetitive intergenic consensus PCR assays. These strains were also indistinguishable with a previously described PCR assay that distinguished the citrus strains from all other strains of Xylella fastidiosa. Because we were not able to document any genomic diversity in our collection of Xylella fastidiosa strains isolated from diseased citrus, the observed gradient of increasing disease severity from southern to northern regions of S?o Paulo State is unlikely due to the presence of significantly different strains of the pathogen in the different regions. When comparisons were made to reference strains of Xylella fastidiosa isolated from other hosts using these methods, four groups were consistently identified consistent with the hosts and regions from which the strains originated: citrus and coffee, grapevine and almond, mulberry, and elm, plum, and oak. Independent results from random amplified polymorphic DNA (RAPD) PCR assays were also consistent with these results; however, two of the primers tested in RAPD-PCR were able to distinguish the coffee and citrus strains. Sequence comparisons of a PCR product amplified from all strains of Xylella fastidiosa confirmed the presence of a CfoI polymorphism that can be used to distinguish the citrus strains from all others. The ability to distinguish Xylella fastidiosa strains from citrus and coffee with a PCR-based assay will be useful in epidemiological and etiological studies of this pathogen.     

枸头橙种子中柑桔衰退-茎陷点病毒的鉴定

 采用双抗体夹心-酶联免疫吸附测定试验(DAS-ELISA)对枸头橙种子中柑桔衰退-茎陷点病毒的带毒情况进行了鉴定。结果表明,从严重感染茎陷点病的枸头橙罹病树采集的种子可检测到柑桔衰退病毒(CTV)。种子带毒量以内种皮为较高,剥皮种子次之,外种皮呈阴性。种子在4℃冰箱内贮藏1~2年后仍带毒,但带毒量有所下降。对带毒种子繁殖的实生苗及嫁接在实生苗上的墨西哥来檬的检测结果,有部分呈阳性,种植在防虫隔离网室内的100余株2年生枸头橙实生苗,用无毒珠心系的墨西哥来檬芽嫁接进行生物鉴定,经2年观察未发现症状     

Characterization of Citrus tristeza virus isolates in northern Iran

The biological and molecular properties of four Citrus tristeza virus (CTV) isolates isolated from infected Satsuma trees imported from Japan, and growing in citrus groves in northern Iran (Mahdasht orchards, Mazandaran Province), were investigated. CTV-infected samples were collected from sweet orange trees and grafted onto Alemow (Citrus macrophylla Wester) seedlings. On indicator plants, these isolates produced various symptoms including vein clearing and stem pitting on Mexican lime, Alemow, and Citrus hystrix, and yellowing and stunting on sour orange and grapefruit seedlings. Citrus samples were also surveyed for CTV using serological tests. The coat protein (CP) gene of these isolates was amplified using specific primers, yielding an amplicon of 672 bp for all isolates. Sequence analysis showed 98%–99% sequence homology of Iranian isolates with the Californian CTV severe stem-pitting isolate SY568 and 97%–98% homology with the Japanese seedling yellows isolate NUagA. The Iranian isolates were compared by restriction fragment length polymorphism (RFLP) analysis of the CP amplicon for further classification.     

Populations of Xylella fastidiosa in Plants Required for Transmission by an Efficient Vector

ABSTRACT Xylella fastidiosa, a xylem-limited bacterium that causes Pierce's disease (PD) of grapevine and other diseases, is transmitted efficiently by xylem-feeding leafhoppers. Acquisition of a PD strain of X. fastidiosa by the blue-green sharpshooter (BGSS) from five plant host species-grapevine (Vitis vinifera), Himalayan blackberry (Rubus discolor), California mugwort (Artemisia douglasiana), watergrass (Echinochloa crus-galli), and Bermuda grass (Cynodon dactylon)-was tested at various time intervals after vector inoculation. The minimum incubation periods in plant hosts before BGSS acquired X. fastidiosa were 4, 22, 29, and 25 days for grapevine, blackberry, mugwort, and watergrass, respectively. There were no transmissions by vectors or recoveries of X. fastidiosa by culturing from Bermuda grass in 133 attempts, including 80 attempts with the green sharpshooter, Draeculacephala minerva. The first acquisitions and subsequent transmissions by BGSS occurred after X. fastidiosa multiplied to a population of about 10(4) CFU/g of stem tissue. Higher populations of bacteria in plants resulted in higher rates of transmission. In grapevine, the rate of transmission increased over time (4.5% in the first 10 days to 55% after day 25) as the maximum number of viable CFU of X. fas-tidiosa recovered by culturing also increased (from 5 x 10(5) CFU/g during the first 10 days to 5 x 10(8) after day 25).     

Two Xylella fastidiosa Genotypes Associated with Almond Leaf Scorch Disease on the Same Location in California

ABSTRACT Almond leaf scorch disease (ALSD) has recently reemerged in the San Joaquin Valley of California threatening almond production. ALSD is caused by Xylella fastidiosa, a nutritionally fastidious bacterium. Single nucleotide polymorphisms (SNPs) in the 16S rRNA gene (16S rDNA) of X. fastidiosa strains were identified to characterize the bacterial population in infected trees. Genotype-specific SNPs were used to design primers for multiplex polymerase chain reaction assays of early passage cultures. Two genotypically distinct types of X. fastidiosa strains, G-type and A-type, coexist simultaneously in the same infected almond orchard. This was substantiated by restriction fragment length polymorphism analysis of a different genetic locus, RST31-RST33, which has previously been used to identify and differentiate X. fastidiosa strains. Furthermore, unique bacterial colony morphology was consistently associated with the A-type X. fastidiosa strains. To our knowledge, this is the first report of a mixed genotype infection of X. fastidiosa disease on the same location under natural environmental conditions. The concept of mixed genotype infection could affect the current epidemiological study based on the assumption that one genotype causes ALSD on one location and, therefore, the disease management strategy.     

Molecular characterization of Citrus yellow mosaic badnavirus (CMBV) isolates revealed the presence of two distinct strains infecting citrus in India

Citrus yellow mosaic badnavirus (CMBV) is a non-enveloped, bacilliform DNA virus and the etiologic agent of yellow mosaic disease of citrus in India. The disease was initially reported from the southern parts of India and has now spread to other parts of the country. It is a serious disease of sweet orange (Citrus sinensis) in southern India, where it causes significant yield losses. During a recent survey of citrus groves in the Nagpur region, central India, characteristic mosaic symptoms were observed in mandarin orange (Citrus reticulata) and sweet orange. Virus transmission studies, electron microscopy, PCR amplification and sequencing of cloned PCR products from samples showing mosaic symptoms confirmed the presence of a badnavirus. The CMBV–Nagpur isolate could be transmitted to the Rangpur lime (C. limonia) and acid lime (Citrus aurantifolia) by graft inoculation. Sequence analysis of a segment of ORF-III region and intergenic region (IR) of the viral genome revealed that CMBV–Nagpur isolate formed a distinct clade along with some previously reported isolates that are known to infect acid lime and Rangpur lime. CMBV isolates that infect citrus species other than the acid lime and Rangpur lime formed a second clade. Based on the transmission studies and phylogenetic analyses, it was concluded that at least two strains of CMBV exist in India currently.     

Geographical Genetic Structure of Xylella fastidiosa from Citrus in São Paulo State, Brazil

ABSTRACT A total of 360 Xylella fastidiosa strains were isolated from sweet orange (Citrus sinensis) cv. Pera plants growing in five geographic regions in the Brazilian state of S?o Paulo. The genetic variation of these strains was determined by 15 variable number tandem repeat (VNTR) and 58 random amplified polymorphic DNA (RAPD) markers. The mean values of genetic diversity (H) of X. fastidiosa strains within each geographic region determined by RAPD (H(RAPD)) were substantially lower than H(VNTR) values. H(RAPD) values ranged from 0.00 to 0.095, whereas the H(VNTR) values ranged from 0.024 to 0.285. A highly significant value of Nei's coefficient of gene differentiation (G(ST) = 0.355; P = 0.000) was detected among all five populations. Analysis of the molecular variance (AMOVA) also revealed significant genetic differentiation among regions or populations ( phi(STAT) = 0.810; P< 0.001). In addition, genetic differentiation among subpopulations (plants) within the regions (phi(STAT) = 0.699; P < 0.001) and within each plant (phi(STAT) = 368; P < 0.001) were statistically significant. These high values of genetic differentiation among X. fastidiosa strains from different regions suggest a genetic structure according to region of host origin. However, no apparent correlation between genetic distance and region of origin of populations were supported statistically by Mantel analysis (r = 0.27; P = 0.22).     

柑橘黄龙病菌在染病贡柑枝条和果实橘络内的分布规律

 柑橘黄龙病是柑橘生产上最具毁灭性的病害之一,由目前还无法分离培养的候选韧皮部杆菌亚洲种(“Candidatus Liberibacter asiaticus”, CLas)引起的。早期研究表明CLas在染病柑橘植株内的分布并不均匀。为进一步探究CLas在染病枝条及果实内的空间分布规律,本研究以感染黄龙病的贡柑(带果实)枝条为材料,利用定量PCR(quantitative PCR, qPCR)分析同一枝条不同部位及果实橘络中的CLas浓度。结果表明,带果贡柑枝条不同部位的CLas分布不均匀且以果实橘络部位的CLas浓度最高(15 487.6 CLas cells/ng总DNA)。通过对同一橘络不同片段长度(每1.5 cm)中的CLas进行定量分析发现,同一橘络中的CLas同样呈不均匀分布(CLas个数范围:12 407~10 271 089)。此外,定量分析发现黄龙病引起的畸形果大小两侧橘络中的CLas浓度差异不显著。该结果可为探究CLas在柑橘枝条及果实中的转运规律提供参考。