SCIENTIFIC OPINION. EFSA Panel on Plant Health (PLH) 2, 3

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1 EFSA Journal 2013;11(10):3374 SCIENTIFIC OPINION Statement in response to the comments submitted by the US phytosanitary authorities on a previous EFSA opinion (EFSA Journal 2011;9(12):2461) regarding the export of Florida citrus fruit to the EU 1 EFSA Panel on Plant Health (PLH) 2, 3 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT The European Food Safety Authority (EFSA) has been asked to address the comments submitted in April 2012 by the US phytosanitary authorities on the EFSA scientific opinion on the US request related to the export of Florida citrus fruit. The EFSA Panel on Plant Health acknowledges the responses provided by the United States Department of Agriculture (USDA) and considers that the USDA misinterpreted the conclusions made by the Panel in its previous opinion (EFSA Panel on Plant Health (PLH), 2011). The Panel considers that the extrapolations of the data provided by Shiotani et al. (2009) and Gottwald et al. (2009) were not supported by sound evidence. The Panel considers that both the responses provided by Dr Shiotani and by Dr Gottwald and colleagues do not lead to any major change in the set of scientifically sound information related to citrus canker and Xanthomonas citri pv. citri. The Panel, after evaluating the response provided by the USDA regarding the previous EFSA scientific opinion on citrus canker, considers that the removal of mandatory mitigation measures in field and packinghouse facilities increases the risk of contamination of fruit during the growing season and thus the bacterial load and frequency of infected fruit in consignments. Permitting the trade of citrus fruit originating from infected orchards increases the probability of the presence of X. citri pv. citri on fruit in consignments from those sites. The Panel considers that citrus fruit is a pathway for introduction of X. citri pv. citri into the European Union and the related risk is being evaluated in a separate EFSA opinion under preparation on the plant health risk of citrus canker for the EU. European Food Safety Authority, 2013 KEY WORDS Xanthomonas citri pv. citri, citrus canker, citrus fruit, European Union, USDA-APHIS 1 On request from the European Commission. Question No EFSA-Q , adopted on 18 September Panel members: Richard Baker, Claude Bragard, Thierry Candresse, Gianni Gilioli, Jean-Claude Grégoire, Imre Holb, Michael John Jeger, Olia Evtimova Karadjova, Christer Magnusson, David Makowski, Charles Manceau, Maria Navajas, Trond Rafoss, Vittorio Rossi, Jan Schans, Gritta Schrader, Gregor Urek, Johan Coert van Lenteren, Irene Vloutoglou, Stephan Winter and Wopke van der Werf. Correspondence: plh@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group on Citrus Canker Pest Risk Assessment: Claude Bragard, David Caffier, Charles Manceau, Olivier Pruvost Jan Schans and Christian Vernière, for the preparatory work on this scientific opinion, and EFSA staff: Svetla Kozelska, for the support provided to this scientific opinion. Suggested citation: EFSA PLH Panel (EFSA Panel on Plant Health), Statement in response to the comments submitted by the US phytosanitary authorities on a previous EFSA opinion (EFSA Journal 2011;9(12):2461) regarding the export of Florida citrus fruit to the EU. EFSA Journal 2013;11(10):3374, 63 pp. doi: /j.efsa Available online: European Food Safety Authority, 2013

2 SUMMARY Following a request from the European Commission, the EFSA Panel on Plant Health has been asked to address the comments submitted in April 2012 by the US phytosanitary authorities in response to the recent EFSA opinion on a US request regarding the export of Florida citrus fruit to the EU (EFSA Journal 2011;9(12):2461). The Panel conducted its work taking into account the general principles of the Guidance on a harmonised framework for pest risk assessment and the identification and evaluation of pest risk management options (EFSA Panel on Plant Health (PLH), 2010) and the Guidance on evaluation of risk reduction options (EFSA Panel on Plant Health (PLH), 2012). The opinion has been developed in line with the principles described in the document Guidance of Scientific Committee on transparency in the scientific aspects of risk assessment carried out by EFSA Part 2: General principles (EFSA, 2009). After consideration of the evidence, the Panel reached the following conclusions: The Panel acknowledges the responses provided by the Unites States Department of Agriculture (USDA). Considering the scientific documents from Shiotani et al. (2009) and Gottwald et al. (2009), the USDA misinterpreted the conclusions made by the EFSA PLH Panel in its previous opinion (EFSA Panel on Plant Health (PLH), 2011). The EFSA PLH Panel did not consider those papers to be inconclusive and lack(ing) sound science, but only that the extrapolations of those papers were not supported by evidence. After a detailed review of the response provided by the USDA, the Panel acknowledges that important details that were missing in Shiotani et al. (2009) and Gottwald et al. (2009) are now available from the authors of these scientific papers as personal communications. Nevertheless, the Panel considers that both responses provided by Dr Shiotani and by Dr Gottwald and colleagues do not lead to any major change in the set of scientifically sound information related to citrus canker and Xanthomonas citri pv. citri. The Panel, after evaluating the response provided by the USDA regarding its previous pest risk assessments and risk management analyses, further considers that: removing mandatory mitigation measures in the field increases the risk of contamination of fruit during the growing season and thus the bacterial load and frequency of infected fruit in consignments; removing mandatory mitigation measures in packinghouse facilities contributes to increasing the bacterial load and frequency of infected fruit in consignments; allowing the trade of citrus fruit originating from infected orchards increases the probability of the presence of Xanthomonas citri pv. citri on fruit in consignments from those sites. The Panel considers that citrus fruit is a pathway for the introduction of X. citri pv. citri to the European Union and the related risk is being evaluated in a separate EFSA opinion under preparation on the plant health risk of citrus canker for the EU, addressing another question from the same terms of reference. EFSA Journal 2013;11(10):3374 2

3 TABLE OF CONTENTS Abstract... 1 Summary... 2 Table of contents... 3 Background as provided by the European Commission... 6 Terms of reference as provided by the European Commission... 6 Assessment Introduction Purpose Scope Methodology and data Guidance documents Methods used for analysing and addressing the comments of the US phytosanitary authorities Data and literature search Analysis and EFSA s response to the APHIS response (USDA, 2012) Analysis of answers obtained by USDA from Dr Shiotani on the remarks made by the EFSA PLH Panel on the paper from Shiotani et al. (2009) Dr Shiotani s answer (1) Dr Shiotani s answer (2) Dr Shiotani s answer (3) Dr Shiotani s answer (4) Dr Shiotani s answer (5) Dr Shiotani s answer (6) Dr Shiotani s answer (7) Dr Shiotani s answer (8) Dr Shiotani s answer (9) Dr Shiotani s answer (10) Dr Shiotani s answer (11) Dr Shiotani s answer (12) Dr Shiotani s answer (13) Analysis of answers obtained by USDA from Dr Gottwald, Dr Graham, Dr Bock and Dr Taylor on the remarks made by the EFSA PLH Panel on the paper by Gottwald et al. (2009) Dr Gottwald et al. s answer (1) Dr Gottwald et al. s answer (2) Dr Gottwald et al. s answer (3) Dr Gottwald et al. s answer (4) Dr Gottwald et al. s answer (5) Dr Gottwald et al. s answer (6) Dr Gottwald et al. s answer (7) Dr Gottwald et al. s answer (8) Dr Gottwald et al. s answer (9) Dr Gottwald et al. s answer (10) Dr Gottwald et al. s answer (11) Dr Gottwald et al. s answer (12) Dr Gottwald et al. s answer (13) Dr Gottwald et al. s answer (14) Dr Gottwald et al. s answer (15) Dr Gottwald et al. s answer (16) Dr Gottwald et al. s answer (17) Dr Gottwald et al. s answer (18) Dr Gottwald et al. s answer (19) Dr Gottwald et al. s answer (20) Dr Gottwald et al. s answer (21) EFSA Journal 2013;11(10):3374 3

4 Dr Gottwald et al. s answer (22) Dr Gottwald et al. s answer (23) Dr Gottwald et al. s answer (24) Dr Gottwald et al. s answer (25) Dr Gottwald et al. s answer (26) Dr Gottwald et al. s answer (27) Dr Gottwald et al. s answer (28) Dr Gottwald et al. s answer (29) Dr Gottwald et al. s answer (30) Dr Gottwald et al. s answer (31) Analysis of the comments from USDA on the statements made by the EFSA PLH Panel (EFSA Panel on Plant Health (PLH), 2011) on the PRAs USDA response on the PRAs (1) USDA response on the PRAs (2) USDA response on the PRAs (3) USDA response on the PRAs (4) USDA response on the PRAs (5) USDA response on the PRAs (6) USDA response on the PRAs (7) USDA response on the PRAs (8) USDA response on the PRAs (9) USDA response on the PRAs (10) USDA response on the PRAs (11) USDA response on the PRAs (12) USDA response on the PRAs (13) USDA response on the PRAs (14) USDA response on the PRAs (15) USDA response on the PRAs (16) USDA response on the PRAs (17) USDA response on the PRAs (18) USDA response on the PRAs (19) USDA response on the PRAs (20) USDA response on the PRAs (21) USDA response on the PRAs (22) USDA response on the PRAs (23) USDA response on the PRAs (24) USDA response on the PRAs (25) USDA response on the PRAs (26) USDA response on the PRAs (27) USDA response on the PRAs (28) USDA response on the PRAs (29) USDA response on the PRAs (30) Analysis of the comments from USDA on the statements made by the EFSA PLH Panel (EFSA Panel on Plant Health (PLH), 2011) on the RMAs USDA response on the RMAs (1) USDA response on the RMAs (2) USDA response on the RMAs (3) USDA response on the RMAs (4) USDA response on the RMAs (5) USDA response on the RMAs (6) USDA response on the RMAs (7) USDA response on the RMAs (8) USDA response on the RMAs (9) USDA response on the RMAs (10) USDA response on the RMAs (11) EFSA Journal 2013;11(10):3374 4

5 USDA response on the RMAs (12) USDA response on the RMAs (13) Conclusions Documentation provided to EFSA References EFSA Journal 2013;11(10):3374 5

6 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION The current European Union plant health regime is established by Council Directive 2000/29/EC on protective measures against the introduction into the Community of organisms harmful to plants or plant products and against their spread within the Community (OJ L 169, , p. l). The Directive lays down, amongst others, the technical phytosanitary provisions to be met by plants and plant products and the control checks to be carried out at the place of origin on plants and plant products destined for the Union or to be moved within the Union, the list of harmful organisms whose introduction into or spread within the Union is prohibited and the control measures to be carried out at the outer border of the Union on arrival of plants and plant products. Citrus canker is a serious disease of cultivated citrus plants caused by the strains pathogenic to Citrus of the bacterium Xanthomonas campestris (synonym: Xanthomonas axonopodis pv. citri). Losses due to citrus canker primarily result from defoliation, premature fruit abscission and blemished fruit, which has a reduced market value as fresh fruit. This pathogen is not known to occur in the EU and therefore it is very relevant to prevent its introduction into the EU through appropriate phytosanitary regulation. Xanthomonas campestris (all strains pathogenic to Citrus) is a regulated harmful organism in the EU, listed in Annex IIAI of Council Directive 2000/29/EU. Annexes III; IV AI and VB of this Directive list requirements for the introduction into the EU of citrus plants, including fruits, which could be a pathway for the entry of this pathogen. In addition, temporary emergency are in place which impose additional requirements for the import of certain citrus fruits from Brazil in connection with Xanthomonas campestris (all strains pathogenic to Citrus) (Commission Decision 2004/416/EC; OJ L 151, , p. 76). In spite of the present import requirements against Xanthomonas campestris (all strains pathogenic to Citrus), infested citrus fruit is often intercepted during import inspections. In order to carry out an evaluation of the present EU requirements against Xanthomonas campestris (all strains pathogenic to Citrus), a pest risk analysis covering the whole territory of the EU is needed, which takes into account the latest scientific and technical knowledge for this organism. The work on citrus canker funded by EFSA in the context of the recent Prima Phacie project ( Pest risk assessment for the European Community plant health: A comparative approach with case studies ) is expected to be valuable for the preparation of this pest risk analysis. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION EFSA is requested, pursuant to Article 29(1) and Article 22(5) of Regulation (EC) No 178/2002 4, to provide a pest risk assessment of Xanthomonas campestris (all strains pathogenic to Citrus), to identify risk management options and to evaluate their effectiveness in reducing the risk to plant health posed by this harmful organism. The area to be covered by the requested pest risk assessment is the EU territory. In the risk assessment EFSA is also requested to provide an opinion on the effectiveness of the present EU requirements against Xanthomonas campestris (all strains pathogenic to Citrus), which are listed in Annex III, IV and V of Council Directive 2000/29/EC, as well as in Commission Decision 2004/416/EC and Commission Decision 2006/473/EC, in reducing the risk of introduction of this pest into the EU territory. In addition, guidance on the right denomination of this harmful organism should be included. In its scientific opinion EFSA is requested to address the comments submitted in April 2012 by the US phytosanitary authorities in response to the recent EFSA opinion on a US request regarding the export of Florida citrus fruit to the EU (EFSA Journal 2011;9(2):2461). 4 Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. Official Journal of the European Communities, L 31/1, , p EFSA Journal 2013;11(10):3374 6

7 ASSESSMENT 1. Introduction The United States Department of Agriculture (USDA) (Animal and Plant Health Inspection Service (APHIS), Plant Protection and Quarantine (PPQ), Plant Epidemiology and Risk Analysis Laboratory (PERAL)) reviewed the EFSA Scientific Opinion on the request from the USA regarding export of Florida citrus to the EU (EFSA Panel on Plant Health (PLH), 2011). This review resulted in a 62- page response (USDA, 2012), sent to the European Commission (EC). The core of this USDA response consists of an introduction, plus three general sections (one dedicated to the scientific articles from Shiotani et al. (2009) and Gottwald et al. (2009), one to the pest risk assessments (PRAs) previously made by USDA and one to the risk management analyses (RMAs) previously prepared by USDA) and a conclusion. Four annexes complete this response (answers from Dr Shiotani and Dr Gottwald to the statements made by the EFSA Panel on Plant Health (PLH Panel) in its previous opinion (EFSA Panel on Plant Health (PLH), 2011) and answers from USDA on that EFSA opinion on the related PRAs and RMAs). Basically the USDA considers in its document that the central point of disagreement ( ) is EFSA s conclusion that a conceptually possible pathway justifies phytosanitary measures when scientific evidence demonstrates that transmission of the disease from fruit requires highly contrived experimental conditions that are far beyond any natural scenario or condition that would occur in trade. USDA-APHIS disagrees that the conceptual possibility of a pathway should be interpreted as being equivalent to the probability that such a pathway is feasible and likely to occur under real-world conditions. The abbreviation Xcc used in this document refers to Xanthomonas citri pv. citri (synonyms: Xanthomonas citri subsp. citri, Xanthomonas axonopodis pv. citri) Purpose The pest risk assessment and the identification and evaluation of risk management options, which are requested in the first part of the terms of reference, are being addressed in a separate EFSA opinion under preparation. This document presents an analysis and the EFSA s reply to the comments submitted in April 2012 by the US phytosanitary authorities. These comments aimed to respond to the recent EFSA opinion on a US request regarding the export of Florida citrus fruit to the EU (EFSA Journal 2011, 9(12):2461) (USDA, 2012), prepared by the EFSA PLH Panel (hereinafter referred to as the Panel) in response to a request from the European Commission Scope The document covers the analysis and the EFSA s reply to the comments submitted in April 2012 by the US phytosanitary authorities in response to the recent EFSA opinion on a US request regarding the export of Florida citrus fruit to the EU (EFSA Journal 2011;9(12):2461) (USDA, 2012). 2. Methodology and data 2.1. Guidance documents The Panel conducted its work taking into account the general principles of the Guidance on a harmonised framework for pest risk assessment and the identification and evaluation of pest risk management options (EFSA Panel on Plant Health (PLH), 2010) and of the Guidance on evaluation of risk reduction options (EFSA Panel on Plant Health (PLH), 2012). The opinion has been developed in line with the principles described in the document Guidance of Scientific Committee on transparency in the scientific aspects of risk assessment carried out by EFSA. Part 2: General EFSA Journal 2013;11(10):3374 7

8 principles (EFSA, 2009). The principles described in this document for risk assessment apply to all the scientific outputs from EFSA Methods used for analysing and addressing the comments of the US phytosanitary authorities In a document dated April 2012 (USDA, 2012), the US phytosanitary authorities provided the EC with responses to the EFSA opinion on the US request regarding the export of Florida citrus fruit to the EU (EFSA Journal 2011,9(12):2461). That document aims to answer EFSA s comments related to the scientific papers from Shiotani et al. (2009) and Gottwald et al. (2009) used by the USDA and to the USDA PRAs and RMA. Further information was provided and discussed at the Technical meeting with USDA APHIS on citrus canker held on 20 March 2013 (EFSA, 2013). For transparency, clarity and comprehensiveness, all original statements by the EFSA PLH Panel (EFSA Panel on Plant Health (PLH), 2011) and all answers from the USDA (USDA, 2012) are taken up and addressed one after the other. For clarity and transparency, the Panel will deal in the first part with the explanations given by Dr Shiotani, then in the second part with those given by Dr Gottwald and colleagues. In a third part, the Panel deals with the comments related to the USDA PRAs and finally in a fourth part with those dealing with the RMAs Data and literature search The Panel made use of the extensive bibliographic collection on citrus canker already gathered for the EFSA opinions in 2006 and 2011 and focused the literature search on publications that had appeared since then. Literature searches were performed consulting several sources such as the ISI Web of Knowledge database including Web of Science, Current Content Connect, CABI CAB Abstracts, Food Science and Technology Abstracts and Journal Citation Reports. Searches on the Internet were also carried out. The sources of all data used by the Panel in this opinion are listed in the reference section. The following references Workshop Transcript, 2000; Nat. Plant Board, 2002; Anonymous, 2005a, b were used by the USDA in their response (USDA, 2012) without providing the full reference. These citations are marked in the text with an asterisk (*). Owing to the need for exact citations in the quoted text not all references correspond exactly to the references as provided in the reference section of this opinion. The exact specification of these references according to the reference section of this opinion is added in superscript font to the original reference in the text. 3. Analysis and EFSA s response to the APHIS response (USDA, 2012) 3.1. Analysis of answers obtained by USDA from Dr Shiotani on the remarks made by the EFSA PLH Panel on the paper from Shiotani et al. (2009) Dr Shiotani s answer (1) Original EFSA comment: The PLH Panel considers that the technique used in the 2005 studies to extract the Xanthomonas citri subsp. citri cells from fruit rinds to be used as templates for PCR [polymerase chain reaction] is not suitable. Bacterial cells do not concentrate in the pellet by centrifugation at only g for 10 min; at least g must be applied. It is not possible to determine the occurrence of Xanthomonas citri subsp. citri on fruit using this protocol. As a minimum, testing the rate of recovery of bacteria from suspensions at different concentrations would have been desirable to evaluate the efficiency of this procedure in recovering bacteria. Response from Dr Shiotani: I always apply the gravity and holding time (1 500 g for 10 min) to concentrate the transformed competent cells of Xcc by electroporation just before plating them on a selective medium. The rate of recovery of Xcc cells by centrifugation at EFSA Journal 2013;11(10):3374 8

9 1 500 g for 10 min was 60 % approximately when the initial concentration of the bacterial suspension was 10 3 cfu/ml. Thus, we consider that this condition is enough to concentrate the bacterial cells. Response from the PLH Panel: Analytical processes have to be adapted to the objective of the experiment and proof of their efficiency shall be carefully documented. Working with pure cultures for electroporation and concentrating bacterial cells from infected plant material cannot be compared. The Panel maintains that the described procedure is most probably not adapted and that, at least and as a minimum, testing the rate of recovery of bacteria at different concentrations would have been desirable to evaluate the efficiency of this procedure in recovering bacteria. There is a risk of discarding the target bacteria and thereby obtaining false-negative results Dr Shiotani s answer (2) Original EFSA comment: The PCR procedure used by the authors is that developed by Hartung et al. (1993). This PCR (primer pair 2 3) produced a sensitivity of 10 3 cfu ml 1 (Hartung et al. 1993) to 10 2 cfu ml 1 from pure cultures (Golmohammadi et al., 2007). To obtain similar sensitivity in citrus fruit, Golmohammadi et al. (2007) indicated that a DNA extraction was required before amplification but no indication of this step is given by Shiotani et al. (2009). The authors present neither a standardization method to evaluate the sensitivity in their conditions (e.g. a dilution series with or without fruit tissues) nor the use of a positive control to provide a basis for interpreting the PCR results. In addition, PCR inhibitors are usually released from rinds of citrus fruit and any negative effect on the PCR sensitivity should have been discounted based on preliminary trials. The amount of Taq polymerase utilised in the amplification protocol (0.5 U Ampli Taq per reaction mixture) was half of that used by Golmohammadi et al. (2007) and this could also have played a role in obtaining a lower sensitivity in the PCR reactions. At that time (i.e. 2006), real-time PCR procedures had already been developed to detect Xanthomonas citri subsp. citri and were shown to be more sensitive (Mavrodieva et al., 2004; Golmohammadi et al., 2007). Those procedures were not used in this study even though the detection of the pathogen was the key point in answering the objectives. Response from Dr Shiotani: We used the PCR procedure developed by Hartung et al. (1993). We used positive controls for each the experiment and confirmed the sensitivity of this method. It appears to be probable that real-time PCR procedures are more sensitive. However, I doubt if the detection of Xcc by PCR procedure always indicates the existence of the living cells in the samples determined. Response from the PLH Panel: The aim of the comment of the EFSA PLH Panel was not to reject PCR and to replace it by real-time PCR. Real-time PCR just shows some advantages over PCR, but PCR remains a good tool when used with all the necessary precautions. The Panel acknowledges that Shiotani et al. (2009) used positive controls and confirmed sensitivity, but no factual element is given in the paper. PCR remains a technique of choice. Nevertheless, its use with natural samples often leads to difficulties that dramatically reduce its sensitivity. Various positive controls are required to prove that the experiments carried out are appropriate to reach the objectives. The potential difficulties given in the EFSA opinion (EFSA Panel on Plant Health (PLH), 2011), e.g. absence of DNA extraction, proof of appropriate control of inhibitors, etc. are not addressed and the concerns raised by the EFSA PLH Panel remain. EFSA Journal 2013;11(10):3374 9

10 Dr Shiotani s answer (3) Original EFSA comment: Pathogenicity testing was done by infiltration into mature attached leaves of Navel oranges. The PLH Panel considers that mature leaves are known to be less susceptible than young leaves (Gottwald and Graham, 1992; Vernière et al., 2003) and therefore, they are less appropriate to detect low levels of bacteria, as would be expected on asymptomatic or symptomatic Satsuma fruit. Similarly, the detection level of this bioassay was not evaluated and it is not possible to interpret a null detection. Gottwald and Graham (1992) showed that an inoculum concentration of 10 4 cfu ml 1 in 200 μl was necessary to produce lesions on young susceptible leaves of grapefruit without wounding. In Shiotani et al. (2009) studies, a 30 μl aliquot of inoculum was used for the bioassays on mature sweet orange leaves, but the concentration of this inoculum was not provided. Response from Dr Shiotani: As referred in our article in 2009, Goto et al. (1970) suggested that infiltrating detection technique could detect one cell of Xcc. I have confirmed the excellent sensitivity of this technique to detect Xcc cells even if the bacterial suspension was infiltrated into mature leaves of navel oranges. The technique described by Gottwald and Graham (1992) is quite different from the one we used with regard to the treatment of the plant with the bacterial cells. Therefore, it is inadequate to compare these techniques in ability to produce canker lesions. Response from the PLH Panel: The EFSA PLH Panel acknowledges that Dr Shiotani has confirmed the excellent sensitivity of this technique, but found no detailed reference describing that technique in detail and providing documented proof of its efficacy (i.e. selectivity, sensitivity, reproducibility, robustness, etc.). As Dr Shiotani argues that his technique is different from the one used by Gottwald and Graham (1992), evidence is requested. The Panel recognises that Goto et al. (1970) indicated such a sensitivity threshold using the infiltration detection technique but this work is not cited in Shiotani et al. (2009). In addition, it is not clear in Goto et al. (1970) whether variations in lesion numbers occurred between mature and immature, not fully expanded, leaves after infiltration. Such variations occurred among different citrus species. The Panel still considers that the detection level of this bioassay should have been thoroughly evaluated under the specific conditions of Shiotani s work Dr Shiotani s answer (4) Original EFSA comment: Shiotani et al. (2009) examined Satsuma mandarins from severely infected trees to evaluate if Xanthomonas citri subsp. citri is detectable on the fruit. In 2005 in total 2941 (2 208 asymptomatic, 733 symptomatic) fruits were selected and in 2006 a further (1 283 asymptomatic, 728 symptomatic). The total severity of the disease was expressed by a severity index which showed that in 2006 the disease was more severe than in 2005 (index 18 instead of 7.5). Because no information on the sampling scheme was given, it cannot be evaluated if the data represent typical disease levels in Japan. The severity index is very artificial and gives little information on the existing severity of the infection. Especially the distribution of the observations across the different disease classes is missing. The average number of lesions was not calculated either. Response from Dr Shiotani: Occurrence level of citrus canker disease differs by not only the innate sensitivity of the cultivars to this disease but also environmental conditions, especially, the wind and the rain. Since Satsuma mandarin is moderately resistant to citrus canker, most of orchards growing this cultivar prevent from this threat even though any chemical sprays are not carried out. However, citrus canker disease often occurs in the orchard from which the investigated fruits were harvested in 2005 and 2006, since there are no windbreaks which reduce the severity of the disease. So, the disease level in the orchard is higher among Satsuma mandarin orchards in Japan. The severity index used in the study have EFSA Journal 2013;11(10):

11 been established and proven its validity in the evaluation of the effectiveness of chemicals to citrus canker, which has been conducted by Japan plant Protection Association. Response from the PLH Panel: The Panel can hardly evaluate to what extent the evaluation scheme designed by the Japan Plant Protection Association for the valuation of the effectiveness of chemicals has been adapted to the study done by Shiotani et al. (2009), as no detailed information is provided. Therefore the concerns expressed in the previous EFSA opinion (EFSA Panel on Plant Health (PLH), 2011) remain Dr Shiotani s answer (5) Original EFSA comment: The total sample size is high, but no stratified information on the severity classes is given. To express the statistical uncertainty of the experiment, the 95 % confidence intervals were calculated for the infection rate of Satsuma mandarins with Xanthomonas citri subsp. citri. The upper limit of the confidence interval is 0.10 % in 2005 and 0.15 % in This means, that no observed detections of Xanthomonas citri subsp. citri can not exclude an infection up to these limits. Response from Dr Shiotani: Our research showed that no detectable (and pathogenic) Xcc cell was exuded from not only asymptomatic but also symptomatic Satsuma mandarin fruit harvested from severely infected trees. Therefore, further statistical analysis is not considered. Response from the PLH Panel: The Panel acknowledges the response from Dr Shiotani, but considers that it is necessary to propose completing the results by a statistical analysis of the sampling uncertainty to get a quantification of the limits of detectability in the experiment Dr Shiotani s answer (6) Original EFSA comment: In a second experiment, contaminated and/or infected fruit were put into Navel orange trees as source of inoculum. It was examined if rainwater is a potential means of spreading the bacteria. In this study, the number and selection of examined traps for rainwater is unclear and small (less than 400). The detection limit of sampling beneath the bags with contaminated/infected fruit is unknown. The influence of the amount of rainfall and the dilution effect is unclear. Some detailed information is missing, like time between placement of experimental fruit in the trees/run-off and rainfall or start of rotting. Due to the small sample size, the remaining statistical uncertainty is high. For the various rain events in November 2005 and March 2006, the lack of detection of Xanthomonas citri subsp. citri in the rainwater traps or of symptoms on the leaves only confirm a possible spread below 1.3 % to 3.5 % from all bags with contaminated/infected fruit (upper level of 95 % confidence interval). The sample size in the further experiments was even smaller resulting in less precise results. Response from Dr Shiotani: The traps were shown to be able to catch the Xcc cells although the bacterial strain was not the contaminated Satsuma mandarin fruits. However, we were concerned that the traps under the bags might be insufficient in quantity to catch the Xcc cells from the contaminated fruits without omission. Thus we examined thoroughly citrus canker lesion development among the leaves close to the contaminated fruits. Response from the PLH Panel: The Panel acknowledges that Shiotani et al. are aware of the limits of the experiment they did, but this does not help to answer the concerns raised Dr Shiotani s answer (7) Original EFSA comment: For the experiments conducted in 2005, the authors reported that the plating technique was not suitable to monitor the Xanthomonas citri subsp. citri populations in rinds EFSA Journal 2013;11(10):

12 because of the overgrowth of saprophytic bacterial populations on the semi-selective medium. As a consequence, no conclusion can be drawn on these data. Response from Dr Shiotani: Monitoring the Xcc population in rind was also carried out with PCR technique for the same samples applied to the experiment with plating technique. The investigation with the PCR method showed that any Xcc cells were not detected from the rind wash. Response from the PLH Panel: Dr Shiotani provides here a new piece of information, but, unfortunately, unless results from appropriate controls can be provided, the absence of detection by PCR cannot be attributed to the absence of target bacteria, as saprophytes and other compounds such as copper, common on fruits, may have inhibited the PCR reaction. The Panel confirms that no conclusion can be drawn from these data Dr Shiotani s answer (8) Original EFSA comment: No conclusion can be drawn either on the experiments of 2005 and 2006 on the potential spread of citrus canker disease from Satsuma mandarin fruit. The main concern is related to the use of a rifampicin resistant mutant of Xanthomonas citri subsp. citri (KC21Rif100) that was not previously utilised in Shiotani et al. (2008) and for which no information on citrus fruit colonization, survival or aggressiveness is available. Data on comparative assays using this mutant and a typical wild strain, following their fitness, survival, and virulence on mandarin and orange trees, would be necessary before definitive conclusions could be made. Moreover, the stability of the rifampicin resistance should also have been checked before using the mutant. Response from Dr Shiotani: As described in our article, the resistant strain to rifampicin, KC21Rif100, is a stable, spontaneously derived mutant from strain KC21, which has been shown to be as pathogenic as other Xcc strains in infection studies. Pathogenicity and stability of the resistance to rifampicin of KC21Rif100 were shown in the experiment described in Table 4 of our article. Response from the PLH Panel: Dr Shiotani just states that the KC21Rif100 strain is stable and as pathogenic as other strains, but he does not provide any supporting data. The Panel maintains that information on citrus fruit colonisation, survival or aggressiveness given in the paper from Shiotani et al. (2009) or in any other scientific paper is insufficient. Data on comparative assays using this mutant and a typical wild strain, following their fitness, survival, and virulence on mandarin and orange trees, would be necessary before definitive conclusions could be drawn. Moreover, the stability of the rifampicin resistance and the pathogenicity of rifampicin-resistant strains have not been adequately checked in comparison with wild strains before using the mutant Dr Shiotani s answer (9) Original EFSA comment: The presence of Xanthomonas citri subsp. citri on artificially infected fruit was not monitored before and during the experiment in the groves. It is possible that mature fruit that had been artificially contaminated by soaking showed a decline in populations of Xanthomonas citri subsp. citri but from what starting point is not known. In addition, the bacteria on the surface of the fruit did not survive and were only recovered the day after contamination (Table 5 in Shiotani et al., 2009). The PLH Panel notes that in this case also, the procedures used to recover the bacteria (sonication of the rind followed by centrifugation at g for 10 min) do not seem appropriate. The lack of survival on the surface of Satsuma mandarin fruit may explain the absence of spread observed from fruit contaminated by soaking (Table 3 in Shiotani et al., 2009). EFSA Journal 2013;11(10):

13 Response from Dr Shiotani: As described in Discussion of our article, we speculated that the lack of survival on the surface of Satsuma mandarin surface of Satsuma mandarin fruit may explain the absence of spread observed from fruit contaminated by soaking. Response from the PLH Panel: As this speculation is not supported by any factual data and as the designed protocol does not include any monitoring of Xcc populations, the Panel considers that such a speculation cannot support risk assessment Dr Shiotani s answer (10) Original EFSA comment: During the experiment in the orchard at Kuchinotsu, where attached young Satsuma mandarin fruit had been inoculated by pin-pricking with strain KC21Rif100 (RifR), this strain was recovered three months after inoculation from only three out of 14 lesions at a recovery concentration of cfu per lesion or less (Table 4 in Shiotani et al., 2009). The PLH Panel notes that a variation of the phenotype on Satsuma mandarin fruit has been observed depending on the time of inoculation and has led to different types of symptoms and levels of populations in the lesions (Koizumi, 1972). The authors did not describe which type of lesions was present on the fruit at the beginning of the experiment. Early infection type lesions with a ruptured epidermis or late infection type lesions can maintain and produce different numbers of bacteria. This will influence the dispersal potential of the pathogen present on those fruit lesions. Response from Dr Shiotani: The lesions elicited by strain KC21Rif100 described in our article resembled those of the large type among late infection type named by Koizumi (1972). Response from the PLH Panel: the Panel acknowledges this information and notes that the large type of the late infection type, as described by Koizumi (1972), did not exhibit marked rupture of the epidermis. Even if micro-lesions may exist, this may limit the bacterial exudation from lesions and the recovery of the pathogen from water Dr Shiotani s answer (11) Original EFSA comment: As acknowledged in this paper, and as previously shown (Goto, 1969; Gottwald et al., 1993: Shiotani et al., 2008), Satsuma mandarin (C. unshiu) is a citrus species moderately resistant to resistant to citrus canker. As seen from Fig. 1 in Shiotani et al. (2008), symptoms on leaves developing 40 days after prick inoculation were not erumpent, not really cankerlike, but more pustule-like. However, lesions can slightly rupture the epidermis of the Satsuma mandarin fruit depending on the period of inoculation (Koizumi, 1972). The rupture of epidermis following hypertrophy and hyperplasia in the parenchyma is a major event for the release of Xanthomonas citri subsp. citri bacteria in water (Koizumi, 1976a, b; 1977). This does not occur efficiently with Satsuma mandarin. Differences in the morphology of citrus canker lesions may account for differences in the amount of inoculum released (Timmer et al., 1991). Lesions with few openings and little hyperplasia may be less conducive to a large release of inoculum. Furthermore, the number of bacteria that could be exuded into water from young canker lesions on grapefruit was about 10 4 to 10 5 cfu ml 1 and continued to be exuded at high levels for 24 h (Timmer et al., 1991). Bacteria were found to exude more slowly from older lesions. In addition, the rate of multiplication of Xanthomonas citri subsp. citri on C. unshiu differed significantly from those on C. sinensis (sweet orange, a moderately susceptible to susceptible host) (Shiotani et al., 2008). After 16 days, the bacterial populations decreased on C. unshiu, but not on C. sinensis. At that time the population per leaf lesion was about 10 8 cfu on sweet orange and about 10 6 cfu on Satsuma mandarin. Response from Dr Shiotani: It seems that no additional information for this comment is left. EFSA Journal 2013;11(10):

14 Response from the PLH Panel: The Panel acknowledges that Dr Shiotani does agree with our statement and again points out the limitations of extrapolating the results from the moderately resistant satsuma to other citrus species Dr Shiotani s answer (12) Original EFSA comment: As shown in Table 3 (Shiotani et al., 2009), no leaf lesions were observed in experiments performed in November 2005 and March 2006, probably because the weather conditions were not appropriate for disease development, as stated in the paper. This suggests that conclusions should be drawn from only one experiment in If the results of the experiments shown in Table 2 (Shiotani et al., 2009) were obtained in the same orchards, it is not surprising that bacteria were not collected in rain traps, because the conditions were not favourable for survival and/or because of the use of a mutant with low fitness. As shown in Table 4 (Shiotani et al., 2009), the authors recovered cfu/fruit lesion of the RifR Xanthomonas citri subsp. citri population in three out of 14 lesions from six fruits three months after inoculation, which is not a negligible inoculum source. Response from Dr Shiotani: It is usually so cold in November and March that visible canker lesions are hardly developed from the infection sites. However, Xcc cells can be oozed with rain-water from canker lesions under such the condition. So, it was no wonder that strain KC21Rif100 cells could be scattered with rain-water from the contaminated Satsuma mandarin in March 2005 and November 2006 although the bacterial cells were not actually caught with traps. It is reasonable that the lesions elicited by strain KC21Rif100 on Satsuma mandarin fruits are thought not to be a negligible inoculum source. However, we have no evidence that the bacterial cells were scattered from the contaminated fruits in October The cells of strain KC21Rif100 might be hardly oozed from the lesions, since the lesions produced by the strain slightly ruptured the epidermis of the Satsuma mandarin fruit as described above. Response from the PLH Panel: The Panel acknowledge that Dr Shiotani agrees with the conclusions in the EFSA comment Dr Shiotani s answer (13) Original EFSA comment: The authors state that X. citri pv. citri cannot survive on rotted fruit (Fulton and Bowman, 1929). However, Fulton and Bowman (1929) did not make such a general statement. During their studies, the authors noticed that the development of Penicillium spp. on some of the experimental citrus fruit was followed by a decrease in the number of viable canker bacteria recovered from the edge of the rotted area. However, no decrease in the number of viable bacteria was noticed in the firm areas of the fruit. Response from Dr Shiotani: Under the mild and humid climate condition, the fruits must decompose thoroughly sooner or later. Response from the PLH Panel: The Panel agrees that fruit must decompose thoroughly, sooner or later. One may also state that any bacterium may die sooner or later. Nevertheless, this does not mean that Xcc cannot survive on rotted fruit Analysis of answers obtained by USDA from Dr Gottwald, Dr Graham, Dr Bock and Dr Taylor on the remarks made by the EFSA PLH Panel on the paper by Gottwald et al. (2009) Dr Gottwald et al. s answer (1) Original EFSA comment: Experimental grapefruit fruit were collected in January 2007 in Florida. No data were provided on the environmental conditions, treatments in groves, bacterial populations EFSA Journal 2013;11(10):

15 during the time of fruit collection or on the timing of collection in relation to the harvest period. The PLH Panel notes that Xanthomonas citri subsp. citri bacteria can fluctuate over the year and decrease during winter. Epiphytic populations of Xanthomonas citri subsp. citri recovered from symptomatic leaves fluctuated during the day and were generally higher early in the morning in the presence of dew. The recovery from symptomatic leaves also fluctuated throughout the year and populations recovering seemed to decrease in June July (winter time in Argentina) (Timmer et al., 1996). Xanthomonas citri subsp. citri natural populations in the lesions observed in Argentina did not strongly fluctuate as the lesions aged until the lesions overwintered and then populations decreased about 100 fold (Stall et al., 1980). This decrease can even be drastic through the winter season in Japan (Koizumi, 1977). Thus a discontinuity appears in Xanthomonas citri subsp. citri populations in regions where there is a marked winter season. When the winter temperatures are milder, as in a tropical environment, Xanthomonas citri subsp. citri populations were not strongly affected and decreased approximately by 10 fold (Pruvost et al., 2002). Response from Gottwald et al.: Environmental conditions were those prevalent in commercial citrus orchards in Florida at the time. At this time of year temperatures are still quite high as it is usually still quite warm in Florida, and quite humid with intermittent rain showers. Therefore the fruit were exposed to prime weather for propagation of Xcc. Hence, the fruit were exposed to normal commercial environmental conditions for that year. Fruit were harvested for the experiment at the same time fruit were harvested commercially. This is done to ensure that the fruit we used were exposed to the same environmental conditions and harvest characteristics as commercial fruit destined for export. Bacterial populations at time of harvest were quite high, although as stated in the paper, they were on the decline as we have come to find out is the normal course of bacterial populations as fruit enter into maturity. That is as fruit mature they become less of a compatible environment for the bacteria. As fruit turn color, they actually enter into senescence and the physiological environment within the fruit changes to a less compatible environment for bacterial colonization and survival. Fruit were also harvested in the morning when bacterial populations would have been at their highest. Response from the PLH Panel: The Panel acknowledges the pieces of information provided by Gottwald et al. Nevertheless, no precise climatic data is provided. Information on the environmental conditions is still missing as well as on treatments in groves and on bacterial populations during the time of fruit collection. Considering climatic series available on the Internet (for instance January is the coolest month of the year, with average maximum temperatures between 16 and 24 C and average minimum temperatures between 6 and 18 C, depending on location within Florida. January is also a dry month, with rainfall between 45 and 135 mm (refer for instance to this official website Regarding the import season for grapefruit from the US to the EU, the main period is winter, so the experiment is in line with the export period to the EU Dr Gottwald et al. s answer (2) Original EFSA comment: In Gottwald et al. (2009) studies, collected fruit were treated one day after harvest, but no information was provided on the precise storage conditions of fruit between harvest and application of treatments. The treatment described in the Materials and Methods as (4) pre-wash with water plus detergent followed by chlorine immersion, which, according to the data of Fig. 2A, was the most effective treatment in reducing the number of total bacteria, is mistakenly referred to in the Results as the prewash followed by chlorine and detergent. Response from Gottwald et al.: Fruit were held in the packinghouse in the shade at ambient temperature. These conditions do not appreciably change bacterial populations within lesions. EFSA Journal 2013;11(10):