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

Helvécio Della Coletta-Filho & Sérgio Alves Carvalho &

Luis Fernando Carvalho Silva &

Marcos Antonio Machado

Accepted: 5 March 2014

# Koninklijke Nederlandse Planteziektenkundige Vereniging 2014

Abstract Knowledge about the mechanism of trans-mission of systemic pathogens of citrus species is highlyimportant for the safe movement of citrus germplasm,management of citrus mother trees, and also productionof young plants. Among systemic pathogens of citrus,Xylella fastidiosa the causal agent of the citrus variegat-ed chlorosis (CVC), is one the most important patho-gens causing decline in tree vigour and productivity.Seven-year experiments were conducted to evaluatethe hypothesis of seed-to-seedling transmission ofX. fastidiosa. This bacterium was found colonizing thefruit (exocarp, central axis and mesocarp) and the seedparts (seed coat and endosperm plus embryo). After7 years of PCR assay, no positive PCR detection ofX. fastidiosa was confirmed in seedlings propagatedfrom the seeds infected with X. fastidiosa. This resultdemonstrates the lack of seed-to-seedling transmissionof this bacterium.

Keywords Citrus sinensis . Citrus variegated chlorosis .

Seed-borne pathogen

Transport of plant material accounts for the movementof plant pathogens over great distances which can beresponsible for the emergence of diseases in new areas.Seed-borne bacterial pathogens are of particular concernbecause the management of bacterial diseases is limitedin scope and few measures are effective (Gitaitis andWalcott 2007). Seeds can be contaminated internally byvascular bacteria via the host xylem through the hilumor phloem but infection of the seedlings or transmissionof the pathogen to the seedlings (seed-to-seedling) hasbeen rarely demonstrated (Agarwal and Sinclair 1987).

Xylella fastidiosa (Xf) is a xylem-inhabiting bacteri-um that is able to colonize multiple species of plants,including sweet orange trees (Citrus sinensis L.Osbeck). In this host, the bacterium causes a diseaseknown as citrus variegated chlorosis (CVC), whichresults in serious economic losses as a consequence ofsubstantial decreases in fruit production and quality. Thedisease also causes tree dieback. Long-distance trans-mission of this pathogen results from the movement ofinfected citrus propagation material, and the effective-ness of this pathway explains the rapid spread of CVC tomost of Brazil’s citrus-producing states prior to 2003(Almeida et al. 2013). The vectors that transmit Xf bylocal short-distance or tree-to-tree movement include atotal of 12 currently known leafhopper species (Almeidaet al. 2013). These species are involved in both primaryand secondary forms of disease spread (Laranjeira et al.1998). However, the seed-borne status of Xf has notbeen sufficiently clarified. No evidence of transmissionwas observed by Laranjeira et al. (2000) in an analysisof more than 400 seedlings 60 days after the

Eur J Plant PatholDOI 10.1007/s10658-014-0415-8

H. D. Coletta-Filho : S. A. Carvalho : L. F. C. Silva :M. A. MachadoCentro APTA Citros Sylvio Moreira, IAC,Rod. Anhanguera, km 158, C.P.04, 13490-970 Cordeiropólis,São Paulo, Brazil

H. D. Coletta-Filho (*)Laboratório de Biotecnologia, Centro APTA Citros SylvioMoreira, Instituto Agronômico,Cordeiropólis, Brazile-mail: helvecio@centrodecitricultura.br

germination of seeds collected from diseased fruits.Later, Li et al. (2003) shown that Xf can infect variousparts of fruits and seeds and that almost 23 % of 250seedlings germinated from the infected seeds were pos-itive for Xf 180 days after seedling emergence.However, CVC symptoms, even for the positive plants,could not be confirmed by further study because, ac-cording to the authors, all plants were lost as a result ofstorm damage to the greenhouse.

In view of these findings, the purpose of this studywas to provide conclusive evidence for the hypothesisthat Xf can be transmitted vertically from seeds to seed-lings. Such transmission could have a direct impact oncitrus germplasm exchange, as seeds as well as freshfruit in commerce is a potential pathway for introductionof CVC into CVC-free regions. This information willalso be very important for the management of mothertrees for seed-rootstock production and certificationprograms.

Undersized fruits were collected in April 2000 from7-year-old sweet orange trees cv. ‘Pera’ (Citrus sinensisL. Osbeck) with typical CVC symptoms. Seeds wereremoved from these fruits, washed three times and driedto room temperature. The same procedure was per-formed with the seeds from asymptomatic fruits as acontrol. These seeds were used as a control. Assays todetermine the presence of Xf were run on various tis-sues, such as the exocarp, central column, and mesocarpof both diseased and asymptomatic fruits. Seeds re-moved from infected fruits were divided into six lotsas was done for the fruits. These seeds were either sowninto vermiculite plus pine barks substrate or set aside fordissection (seed coat and endosperm plus embryo) toassay for Xf in each case. The sampled tissues of fruitsand seeds within each of six analyzed lots were mixed toobtain one sample per tissue per lot, totaling 18 samplesfor fruits tissues and 12 for seeds that were submitted toPCR-based assay for detection of Xf. From the sownseeds was determined the percentage of germinatedseedlings per each lot and also the subsequent PCR-based assay for detection of Xf by using leaf petioles ofeach seedling. This procedure was conducted four timesduring a 7-year period. Also, annual inspections tocheck for the symptoms of CVC were done. DNAextraction from all citrus tissues analyzed in this studywas performed based on CTABmethodology accordingto Murray and Thompson (1980) after gridding 200 mgof the citrus fresh tissues (except for seed coat ≈100 mg)in liquid N. PCR amplifications were performed in a

25 μl volume using 2 μl of total DNA (100 ng μl−1) andthe conditions established for the primers RST31/RST33 (Minsavage et al. 1994). The amplicons wereseparated on a 1 % agarose gel. Negative (tissue frommicro-grafted healthy plants grown in a greenhouse)and positive (tissue from symptomatic CVC-infectedplants) controls were used throughout the entireprocedure.

In the present study X. fastidiosa was detected in allof analyzed tissues of the sampled diseased fruits, but indifferent frequencies for exocarp (33 %), central axis(50 %), and mesocarp (33 %). Similar results have beenobserved for many species of vascular bacteria in vari-ous crops (Torres and Maringoni 1997; Xu et al. 2010;Camara et al. 2009; Hilf 2011). Additionally, PCR-positive determinations of Xf were obtained in the seedcoat (67 %) and endosperm (50 %) taken from all lots ofcitrus seeds obtained from symptomatic fruits as alsofound by Li et al. (2003), but at a lower frequency.However, beyond the presence of the pathogen in dif-ferent tissues of seeds and fruits, it is important to obtainconclusive information about the potential transmissionof the bacteria from seeds to seedlings. In the currentstudy, no evidence of seed-to-seedling transmission ofXf was obtained from the analysis of both PCR-baseddiagnosis and CVC symptoms in 412 sweet orangeseedlings grown from six seed lots held in a greenhousefor 7 years (Table 1). Previously, Laranjeira et al. (2000)had also demonstrated the lack of Xf transmission byseeds, but this previous study only analyzed seedlingsduring the first 60 days after germination. Given the

Table 1 Summary of results of seed germination and PCR detec-tion of X. fastidiosa in seeds and seedlings from infected sweetorange during the 7 years of evaluation

Seed lot number Germination (%) Time (months) betweensowing of seed and PCRassaysa

36 48 60 84

1 70.0 0/64 0/64 0/62 0/62

2 55.0 0/76 0/75 0/75 0/75

3 87.0 0/58 0/57 0/57 0/56

4 64.0 0/81 0/81 0/80 0/80

5 77.0 0/62 0/62 0/61 0/61

6 80.0 0/84 0/83 0/82 0/81

Average 72.2

a Number of positive samples/total analyzed

Eur J Plant Pathol

incubation period of the bacterium, which can rangefrom 6 to 12 months after inoculation by a bacterialsuspension (Garcia et al. 2012) or after sharpshooterinfection (Yamamoto et al. 2007), we are confident thata 7-year period would be sufficient to allow Xfmultipli-cation in the plants and detection by traditional PCR, aswell as the expression of symptoms. Based on thisinformation, we can exclude the possibility that falsenegative results were obtained due to a low titre ofbacteria in the plant.

Results in the literature that differ from those of thepresent study were presented by Li et al. (2003), whoreported 23 % Xf citrus seed-to-seedling infection at180 days from germination. However, CVC symptoms,even for the positive plants in that study, could not beconfirmed by further study because, according to theauthors, all plants were lost as a result of storm damageto the greenhouse. Such a high rate of pathogen trans-mission via seeds is considerably greater than the ratesof seed-to-seedling pathogen infection commonly re-ported in the literature. These rates are usually less than1 %, as reported for xylem-inhabiting pathogens such asClavibacter michiganensis subsp. michiganensis in to-mato (Xu et al. 2010) and Xanthomonas campestris pv.campestris in brassica (Roberts et al. 1999).Furthermore, this non-fastidious bacterium was able toinfect the xylem of the seedlings through wounds. Thisroute of infection would be entirely unexpected for Xf, axylem-inhabiting fastidious bacterium.

To our knowledge, seed-to-seedling transmission ofother vascular pathogens in citrus has not been con-firmed to date, even for viruses or viroids.Questionable findings of the seed transmission of citruspsorosis virus have been reported for Poncirus trifoliataand hybrids (Bridges et al. 1965; Carnpiglia et al. 1976),but further tests using more adequate methodology haveyielded negative results (Roistacher 1993; D’Onghiaet al. 2000). In addition, recent studies have shown thatthe phloem vessel bacterium “Candidatus Liberibacterasiaticus” can colonize seeds from HLB-symptomaticfruit but is unable to infect the emerged seedlings(Albrecht and Bowman 2009; Shokrollah et al. 2009;Hartung et al. 2010; Hilf 2011; Graham et al. 2011).

Despite the need for care in the handling of propaga-tive plant material, our research findings that Xf cannotbe transmitted vertically from seeds to seedlings fortu-nately indicate that seed exchange is highly secureagainst possible transmission of this pathogen. Thesefindings also facilitate the management of citrus

rootstock mother trees without a need for protectionwith screens, as is currently required in São PauloState for plants intended for budwood production.

Acknowledgments We thank our lab colleagues and reviewersfor constructive suggestions and discussions. HDCF and MACthanks to CNPq for the scholarship support.

References

Agarwal, V. K., & Sinclair, J. B. (1987). Principles of seedpathology (Vol. 1). Boca Raton: CRC Press.

Albrecht, U., & Bowman, K. D. (2009). Candidatus Liberibacterasiaticus and Huanglongbing effects on citrus seeds andseedlings. HortScience, 44, 1967–1973.

Almeida, R. P. P., Coletta-Filho, H. D., & Lopes, J. R. S. (2013).Xylella fastidiosa–Citrus variegated chlorosis. In D. Liu(Ed.), Manual of security sensitive microbes and toxins.Taylor & Francis Group, USA. (in press).

Bridges, G. D., Youtsey, C. O., & Nixon, R. R. (1965).Observation indicating psrosis transmission by seed ofCarrizo citrange. Proccedings of the Florida StateHorticultural Society, 78, 48–50.

Camara, R. C., Vigo, S. C., & Maringoni, A. C. (2009). Plant-to-seed transmission of Curtobacterium flaccumfaciens pv.flaccumaciens in a dry bean cultivar. Journal of PlantPathology, 91, 549–554.

Carnpiglia, H. G., Silveira, C.M., & Salibe, A. A. (1976). Psorosistransmission through seeds of trifoliate orange. Proceedingsof the 7th Conference of International Organization of CitrusVirologists, 132–134. Riverside, CA.

D’Onghia, A. M., Djelouah, K., & Savino, V. (2000). Serologicaldetection of Citrus psorosis virus in seeds but not in seedlingsof infected mandarin and sour orange. Journal of PlantPathology, 82, 233–235.

Garcia, A. L., Torres, S. C. Z., Heredia, M., & Lopes, S. A. (2012).Citrus responses to Xylella fastidiosa infection. PlantDisease, 96, 1245–1249.

Gitaitis, R., &Walcott, R. (2007). The epidemiology and manage-ment of seedborne bacterial diseases. Annual Review ofPhytopathology, 45, 371–397.

Graham, J. H., Johnson, E. G., Bright, D. B., & Irey, M. S. (2011).Lack of development of Huanglongbing in seedlings fromcitrus seed. Proccedings of the Florida State HorticulturalSociety, 124, 79–84.

Hartung, J. S., Halbert, S. E., Pelz-Stelinski, K., Brlansky, R. H.,Chen, C., & Gmitter, F. G. (2010). Lack of evidence fortransmission of ‘Candidatus Liberibacter asiaticus’ throughcitrus seed taken from affected fruit. Plant Disease, 94,1200–1205.

Hilf, M. E. (2011). Colonization of citrus seed coats by‘Candidatus Liberibacter asiaticus’: implications for seedtransmission of the bacterium. Phytopathology, 101, 1242–1250.

Eur J Plant Pathol

Laranjeira, F. F., Bergamin, F. A., & Amorim, L. (1998).Dynamics and structure of citrus variegated chlorosis(CVC) foci. Fitopatologia Brasileira, 23, 36–41.

Laranjeira, F. F., Pompeu, J., Jr., & Palazzo, D. (2000). Sementesde frutos de laranjeira ‘Natal’ afetados pela clorose variegadados citros: germinação, crescimento de plântulas e nãotransmissão de Xylella fastidiosa. Laranja, 1, 161–173 (inPortuguese).

Li, W. B., Pria Júnior, W. D., Lacava, P. M., Qin, X., & Hartung, J.S. (2003). Presence of Xylella fastidiosa in sweet orange fruitand seeds and its transmission to seedlings. Phytopathology,93, 953–958.

Minsavage, G. V., Thompson, C. M., Hopkins, D. L., Leite, R. M.V. B. C., & Stall, R. E. (1994). Development of a polymerasechain-reaction protocol for detection of Xylella fastidiosa inplant tissue. Phytopathology, 84, 456–461.

Murray,M. G., & Thompson,W. F. (1980). Rapid isolation of highmolecular weight plant DNA. Nucleic Acids Research, 8,4321–4325.

Roberts, S. J., Hiltunen, L. H., Hunter, P. J., & Brough, J. (1999).Transmission from seed to seedlings and secondary spread ofXanthomonas campestris pv. campestris in Brassica

transplants: effects of dose and watering regime. EuropeanJournal of Plant Pathology, 105, 879–889.

Roistacher, C. N. (1993). Psorosis—A review. Proceedings of the12th Conference of International Organization of CitrusVirologists, 139–154. Riverside, CA.

Shokrollah, H., Abdullah, T. L., Sijam, K., & Abdullah, S. N. A.(2009). Determination of the presence of huanglongbingin seeds and movement of the pathogen in Citrusreticulata. American Journal of Applied Sciences, 6,1180–1185.

Torres, J. P., & Maringoni, A. C. (1997). Reaction of dry bean toXanthomonas campestris pv. phaseoli and seed transmissionof the bacterium. Fitopatologia Brasileira, 22, 546–549.

Xu, X., Miller, S. A., Baysal-Gurel, F., Gartemann, K.-H.,Eichenlaub, R., & Rajashekara, G. (2010). Bioluminescenceimaging of Clavibacter michiganensis subsp. michiganensisinfection of tomato seeds and plants. Applied andEnvironmental Microbiology, 76, 3978–3988.

Yamamoto, P. T., Felippe, M. R., Caetano, A. C., Sanches, A. L.,& Lopes, J. R. S. (2007). First report of Fingeriana dubiaCavichioli transmitting Xylella fastidiosa to citrus.Fitopatologia Brasileira, 32, 266.

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