First Report of Xanthomonas citri pv. citri-A* Causing Citrus Canker on Lime in Cambodia

Asiatic citrus canker caused by Xanthomonas citri pv. citri (X. citri pv. citri-A) is detrimental to citrus production in tropical and subtropical areas. The bacterium can cause severe infection on many citrus species, initially causing water-soaked leaf lesions that become erumpent and necrotic, often with a chlorotic halo. Severe infection causes premature fruit drop and twig dieback. X. citri pv. citri-A has consequently been subject to eradication and international quarantine regulations. In the 1990s, strains with a host range restricted to Mexican lime (Citrus aurantifolia), but not infecting grapefruit (C. paradisi), were described in different areas of Southwest Asia (4). This variant was designated X. citri pv. citri-A* because of its phenotypic and genetic similarities with X. citri pv. citri. Lime leaves with canker lesions were collected in 2007 from a citrus nursery in Kandal Province, Cambodia and isolations were performed with KC semiselective medium (3). Four Xanthomonas-like strains were further characterized by PCR alongside positive control strain CFBP 2525 from New Zealand. The expected DNA fragment was obtained using primer pair 4/7 (2) from the bacterial strains but not when distilled water was used as a template. Amplified fragment length polymorphism (AFLP) analysis of the four X. citri pv. citri strains from Cambodia and reference strains X. citri pv. citri-A (CFBP 2525, CFBP 2900, LMG 9322), -A* (CFBP 2911, JF90-2, JK2-10, JK143-1, JM47-2), and X. citri pv. aurantifolii (CFBP 2866, CFBP 2868, CFBP 2901) using SacI/MspI and four primer pairs (1) separated the Cambodian strains into two distinct haplotypes (i.e., AFLP fingerprint patterns). One haplotype was closely related (evolutionary genome divergences [EGD] ≤0.006 [1]) to X. citri pv. citri-A strains with a wide host range and the other was most genetically related to a strain of X. citri pv. citri-A* from Thailand (EGD of 0.003). On the basis of AFLP, the Cambodian isolates were not related to X. citri pv. aurantifolii (EGD values of >0.060). When inoculated to Mexican lime and Duncan grapefruit using a detached leaf assay in which inoculum droplets containing ∼1 × 106 CFU were deposited on wounds (4), the strains genetically related to X. citri pv. citri-A produced typical canker lesions on both citrus species a week after inoculation, whereas the Cambodian strains related to X. citri pv. citri-A* by AFLP analysis only produced canker lesions on lime. Our finding extended the geographical distribution of pathotype A*. Identification of both pathotypes from a few samples collected in a nursery suggests a potential for large-scale distribution of these strains within the citrus orchards in Cambodia, where the most important citrus crop is sweet orange, suggesting that the occurrence of X. citri pv. citri-A* is of moderate economic significance, in contrast with X. citri pv. citri-A strains with a wide host range. Diseased citrus nursery plants are a major source of primary inoculum in developing countries. Sanitation of citrus nurseries against citrus canker in Cambodia is a prerequisite for improved management of the disease. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) J. S. Hartung et al. Phytopathology 86:95, 1996. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) C. Vernière et al. Eur. J. Plant Pathol. 104:477, 1998.

Download Full-text

First Report in Thailand of Xanthomonas axonopodis pv. citri-A* Causing Citrus Canker on Lime

Plant Disease ◽

10.1094/pdis-91-6-0771a ◽

2007 ◽

Vol 91 (6) ◽

pp. 771-771 ◽

Cited By ~ 8

Author(s):

L. Bui Thi Ngoc ◽

C. Vernière ◽

O. Pruvost ◽

N. Kositcharoenkul ◽

S. Phawichit

Keyword(s):

Host Range ◽

Severe Infection ◽

Citrus Canker ◽

Aflp Analysis ◽

Bacterial Disease ◽

Southwest Asia ◽

Citrus Species ◽

Xanthomonas Axonopodis ◽

Mexican Lime ◽

Wide Range

Asiatic citrus canker, caused by Xanthomonas axonopodis pv. citri (Xac-A), is a bacterial disease of economic importance in tropical and subtropical citrus-producing areas. Xac-A can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with a water-soaked margin. Severe attacks cause premature fruit drop and twig dieback. It has consequently been submitted to eradication efforts and international regulations. Recently, a group of strains with a host range restricted to Mexican lime (Citrus aurantifolia), but not infecting grapefruit (C. paradisi) known to be very susceptible to Xac-A, was described in different areas of southwest Asia, including Saudi Arabia, Oman, Iran, and India (3). Phenotypic and genetic similarities with Xac-A designated it as a variant called X. axonopodis pv. citri-A*. A variant with a similar restricted host range, designated X. axonopodis pv. citri-Aw, was also detected in Florida and likely originated from India (2). Amplified fragment length polymorphism (AFLP) analysis of 26 isolates from Thailand and additional reference isolates from Xac-A, -A*, -Aw, and X. axonopodis pv. aurantifolii (2,3) using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5′-labeled SacI + C primer for the selective amplification step), separated the Thai isolates into two distinct groups. A group composed of 20 isolates from different citrus species, including five isolates from lime, was closely related to strains with a large host range and previously identified as Xac-A. A second group, solely composed of six isolates from lime, was genetically related to Xac-A*. All Thai isolates were collected before 1991. Isolates genetically closed to Xac-A* originated from the central and northern provinces, whereas isolates related to Xac-A originated from all sampled provinces. On the basis of AFLP, no Thai isolate was related to X. axonopodis pv. aurantifolii. A specific X. axonopodis pv. citri nested-PCR assay (1) produced the expected fragments for all Thai isolates. Mexican lime leaves inoculated with Thai isolates of Xac-A and Xac-A* using a detached leaf assay (3) showed typical canker symptoms 1 week after inoculation. When inoculated to grapefruit or sweet orange, the Thai isolates genetically related to Xac-A* by AFLP analysis did not induce any canker symptoms, while isolates related to Xac-A produced canker symptoms on these two citrus species. In Thailand, Xac-A* induced severe symptoms on lime trees, including extensive defoliation and numerous twig cankers that often developed as diebacks. The Xac-A* variant appears epidemiologically important on lime, consistent with previous reports from southwest Asia. The detection of Xac-A* in Thailand makes it necessary to evaluate its geographic distribution in southeast Asia. References: (1) J. S. Hartung et al. Phytopathology 86:95, 1996. (2) X. A. Sun et al. Plant Dis. 88:1179, 2004. (3) C. Vernière et al. Eur. J. Plant Pathol. 104:477, 1998.

Download Full-text

Pathotype Identification of Xanthomonas citri pv. citri Strains Causing Citrus Canker in Vietnam

Plant Disease ◽

10.1094/pdis-93-6-0671a ◽

2009 ◽

Vol 93 (6) ◽

pp. 671-671 ◽

Cited By ~ 3

Author(s):

L. Bui Thi Ngoc ◽

C. Vernière ◽

C. Boyer ◽

K. Vital ◽

O. Pruvost ◽

...

Keyword(s):

Host Range ◽

Citrus Canker ◽

Pcr Analysis ◽

Aflp Analysis ◽

Bacterial Disease ◽

Citrus Paradisi ◽

Citrus Species ◽

Xanthomonas Citri ◽

Mexican Lime ◽

Wide Range

Asiatic citrus canker caused by Xanthomonas citri pv. citri is a bacterial disease of major economic importance in tropical and subtropical citrus-producing areas. It probably originated in Asia (2). X. citri pv. citri induces erumpent, callus-like lesions with a water-soaked margin on aerial organs of the plants. Severe attacks cause premature fruit drop and twig dieback. This pathogen has consequently been subjected to international quarantine regulation and eradication efforts. Two pathogenic variants of X. citri pv. citri can be separated by their host range. X. citri pv. citri pathotype A strains cause severe infection worldwide in a wide range of citrus species; grapefruit (Citrus paradisi) is particularly susceptible. More recently, another group of strains from different areas of West Asia has been designated as X. citri pv. citri pathotype A* (4). These A* strains are genetically related to X. citri pv. citri, but their host range is primarily restricted to Mexican lime (C. aurantifolia) and they do not infect grapefruit. Strains similar in host range were later reported in Florida, Thailand, and Cambodia (2). In this study, we investigated the distribution of X. citri pv. citri pathotypes in Southeast Asia. A large survey on citrus was conducted in 14 provinces in the north (Ha Noi, Hung Yen, Nghe Han, Ha Ting, and Phu Tho) and south (Can Tho, Long An, Dong Nai, Tien Giang, Vinh Long, Ben Tre, Dong Thap, Vung Tau, and Lam Dong) of Vietnam. We collected 557 X. citri pv. citri isolates, after cultivation on KC semiselective medium (3), from citrus species, including 60 strains from Mexican lime in eight provinces. Ligation mediated (IS-LM)-PCR analysis using primers targeting three insertion sequences (1) was done on all Vietnamese strains and on additional reference strains of X. citri pv. citri-A, -A*, and X. citri pv. aurantifolii. IS-LM-PCR indicated that all Vietnamese isolates were pathotype A and did not include any with a restricted host range (X. citri pv. citri-A* and X. citri pv. aurantifolii). Amplified fragment length polymorphism (AFLP) analysis was carried out on a subset of 84 X. citri pv. citri strains, including 22 strains from Mexican lime from seven provinces. AFLP was carried out using SacI/MspI and four primer pairs (unlabeled MspI +1 [A, C, T or G] primers and 5′-labeled – SacI + C primer for the selective amplification step) (2) and the data confirmed that all Vietnamese X. citri pv. citri strains were genetically related to pathotype A strains. Mexican lime and Duncan grapefruit or pineapple sweet orange leaves were inoculated with 25 strains from lime (representative of the genetic diversity) using a detached leaf assay (3) and they produced typical canker lesions on both host species. In spite of the presence of pathotype A* strains in neighboring countries (2), no strains genetically or pathogenically related to this pathotype were identified in this collection. A survey of commercial Mexican lime orchards, especially in Vietnamese provinces bordering Cambodia, should be undertaken to detect and eradicate A* strains because these are known to strongly impact lime production in other parts of Asia (e.g., Thailand). References: (1) L. Bui Thi Ngoc et al. Appl. Environ. Microbiol. 75:1173, 2009. (2) L. Bui Thi Ngoc et al. FEMS Microbiol. Lett. 288:33, 2008. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) C. Vernière et al. Eur. J. Plant Pathol. 104:477, 1998.

Download Full-text

First Report of Xanthomonas citri pv. citri-A* Causing Citrus Canker on Lime in Ethiopia

Plant Disease ◽

10.1094/pdis-93-2-0203b ◽

2009 ◽

Vol 93 (2) ◽

pp. 203-203 ◽

Cited By ~ 9

Author(s):

E. Derso ◽

C. Vernière ◽

O. Pruvost

Keyword(s):

Host Range ◽

Rift Valley ◽

Disease Incidence ◽

Citrus Canker ◽

Aflp Analysis ◽

Xanthomonas Citri ◽

First Report ◽

Mexican Lime ◽

Wide Range ◽

Citrus Production

Asiatic citrus canker caused by Xanthomonas citri pv. citri hinders national citrus markets in tropical and subtropical areas and international trade. The bacterium induces erumpent, callus-like lesions causing defoliation, premature fruit drop, and twig dieback. Because of the damage caused by infection and reduced marketability of fruit, several countries have undergone eradication. Strains with different host ranges have been described. Pathotype A strains are the most widespread and produce canker in a wide range of citrus species. Pathotype A* strains with a host range restricted to Mexican lime (Citrus aurantifolia), Tahiti lime (C. latifolia), and alemow (C. macrophylla), but not infecting the susceptible species grapefruit (C. paradisi), were described in different areas of Asia (4). Reemergence of X. citri pv. citri pathotype A was recently described in Africa as affecting citrus production in Mali and Somalia. Canker-like infected citrus trees with symptoms on leaves, fruits, and stems were first observed in 2004 in Ethiopia in the Rift Valley Region. After a survey conducted in 2008, the disease was recorded in different areas of the Rift Valley located in the lowlands (altitude <1300 m, daily mean temperatures 24 to 29°C) and confirmed to only affect Mexican lime orchards with disease incidence as much as 80%. Ten canker-like infected leaves were collected during this survey from eight different orchards distributed along the infected area. Isolations were performed using KC semiselective medium (3), and Xanthomonas-like isolates were further characterized. PCR was used to check the identity of these isolates by using X. citri pv. citri strain CFBP 2525 from New Zealand as the positive control and distilled water as the template for the negative control. The DNA fragment typical of X. citri pv. citri was obtained from all the bacterial isolates using the diagnostic primer pair 4/7 (2). Amplified fragment length polymorphism (AFLP) analysis of the 80 Ethiopian isolates and additional reference isolates from X. citri pv. citri-A, -A*, and pv. aurantifolii using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5′-labeled-SacI + C primer for the selective amplification step) (1) grouped all the Ethiopian isolates in a cluster that was comprised of only X. citri pv. citri pathotype A* strains. On the basis of the AFLP, Ethiopian isolates were only distantly related to X. citri pv. aurantifolii. When inoculated to Mexican lime and Duncan grapefruit by a detached leaf assay (4), all of the Ethiopian strains produced canker on lime only. This confirms the larger geographical distribution of pathotype A*, and to our knowledge, is the first report of its presence on the African continent. This could allow studying the epidemiology of pathotype A* strains in a unique situation where they do not compete with pathotype A strains. The molecular characterization of Ethiopian strains suggests that this introduction event is not related to the recent introduction of citrus canker in neighboring Somalia where X. citri pv. citri pathotype A was identified. Ethiopia will have to prevent the introduction of this wide host range pathotype to avoid further negative impacts on citrus production. References: (1) N. Ah-You et al. Phytopathology 97:1568, 2007. (2) J. S. Hartung et al. Phytopathology 86:95, 1996. (3) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (4) C. Vernière et al. Eur. J. Plant Pathol. 104:477, 1998.

Download Full-text

From Local Surveys to Global Surveillance: Three High-Throughput Genotyping Methods for Epidemiological Monitoring of Xanthomonas citri pv. citri Pathotypes

Applied and Environmental Microbiology ◽

10.1128/aem.02245-08 ◽

2008 ◽

Vol 75 (4) ◽

pp. 1173-1184 ◽

Cited By ~ 34

Author(s):

Lan Bui Thi Ngoc ◽

Christian Verniï¿½re ◽

Philippe Jarne ◽

Sylvain Brisse ◽

Fabien Guï¿½rin ◽

...

Keyword(s):

Host Range ◽

Phylogenetic Signal ◽

Variable Number Tandem Repeat ◽

Citrus Canker ◽

Variable Number ◽

Aflp Analysis ◽

Xanthomonas Citri ◽

Epidemiological Monitoring ◽

Or Groups ◽

Strain Collection

ABSTRACT Asiatic citrus canker is a major disease worldwide, and its causal agent, Xanthomonas citri pv. citri, is listed as a quarantine organism in many countries. Analysis of the molecular epidemiology of this bacterium is hindered by a lack of molecular typing techniques suitable for surveillance and outbreak investigation. We report a comparative evaluation of three typing techniques, amplified fragment length polymorphism (AFLP) analysis, insertion sequence ligation-mediated PCR (IS-LM-PCR) typing, and multilocus variable-number tandem-repeat analysis (MLVA), with 234 strains originating from Asia, the likely center of origin of the pathogen, and reference strains of pathotypes A, A*, and Aw, which differ in host range. The typing techniques were congruent in describing the diversity of this strain collection, suggesting that the evolution pattern of the bacterium may be clonal. Based on a hierarchical analysis of molecular variance, the AFLP method best described the genetic variation found among pathotypes whereas MLVA best described the variation found among individual strains from the same countries or groups of neighboring countries. IS-LM-PCR data suggested that the transposition of insertion sequences in the genome of X. citri pv. citri occurs rarely enough not to disturb the phylogenetic signal. This technique may be useful for the global surveillance of non-epidemiologically related strains. Although pathological characteristics of strains could be most often predicted from genotyping data, we report the occurrence in the Indian peninsula of strains genetically related to pathotype A* strains but with a host range similar to that of pathotype A, which makes the classification of this bacterium even more complicated.

Download Full-text

First Report of Xanthomonas citri pv. citri Causing Citrus Canker in Mali

Plant Disease ◽

10.1094/pdis-92-6-0977b ◽

2008 ◽

Vol 92 (6) ◽

pp. 977-977 ◽

Cited By ~ 8

Author(s):

Y. N. Traoré ◽

L. Bui Thi Ngoc ◽

C. Vernière ◽

O. Pruvost

Keyword(s):

Severe Infection ◽

Citrus Canker ◽

The United States ◽

Positive Control ◽

Aflp Analysis ◽

Bacterial Disease ◽

African Continent ◽

Xanthomonas Citri ◽

Epidemic Area ◽

Wide Range

Citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of economic importance in tropical and subtropical citrus-producing areas. X. citri pv. citri can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with water-soaked margins leading to premature fruit drop and twig dieback. It has consequently been subjected to eradication efforts and international regulations. Citrus canker occurs in Asia, South America, the United States, parts of Oceania, and some islands off the African continent (Comoros, Mauritius, Reunion, and Seychelles islands). It was described on the African continent, but in some cases, diagnosis errors might have occurred. The only well-documented outbreak occurred in South Africa where it was eradicated at the beginning of the twentieth century. In 2004, citrus canker symptoms on limes, sweet oranges, tangerines, and sour oranges were reported from different orchards around Bamako and in the Koulikoro Province of Mali. Isolations were performed on KC semiselective medium (2). PCR was used to check the identity of the pathogen, testing 21 Xanthomonas-like strains collected from citrus in the epidemic area. X. citri pv. citri strain CFBP 2525 from New Zealand was also used as the positive control, and the expected DNA fragment was obtained from all the isolates using primer pair 4/7 (1), whereas no fragment was observed for negative controls (distilled water as the template). Amplified fragment length polymorphism (AFLP) analysis of these X. citri pv. citri strains from Mali and other reference strains from X. citri pv. citri-A, -A*, and X. axonopodis pv. aurantifolii (3), using SacI/MspI and four primer pairs (unlabeled MspI + 1 [A, C, T, or G] primers and 5′-labeled-SacI + C primer for the selective amplification step), showed that the strains were closely related to X. citri pv. citri-A strains with a wide host range. On the basis of AFLP, the Mali strains were not closely related to X. axonopodis pv. aurantifolii. One week after inoculation, Duncan grapefruit and alemow citrus leaves inoculated with all strains from Mali by a detached leaf assay (3) developed erumpent, callus-like tissue at wound sites, similar to the reaction produced by X. citri pv. citri strain CFBP 2525 (positive control). A survey conducted in 2006 in nine orchards revealed disease incidences of 50, 15, 24, and 25% for lime, sweet orange, sour orange, and tangor groves, respectively. As of this report, citrus canker has spread to new citrus orchards and this might be due to the propagation and dissemination of infected material from small nurseries. To our knowledge, this represents the first outbreak of citrus canker in West Africa. Spread of the pathogen in Mali and neighboring countries should be monitored and a drastic surveillance of citrus nurseries in the region should be performed. References: (1) J. S. Hartung et al. Phytopathology 86:95, 1996. (2) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005. (3) C. Vernière et al. Eur. J. Plant Pathol. 104:477, 1998.

Download Full-text

First Report of Xanthomonas citri pv. citri Causing Asiatic Citrus Canker in Burkina Faso

Plant Disease ◽

10.1094/pdis-06-13-0600-pdn ◽

2013 ◽

Vol 97 (12) ◽

pp. 1653-1653 ◽

Cited By ~ 3

Author(s):

C. C. Juhasz ◽

A. Leduc ◽

C. Boyer ◽

F. Guérin ◽

C. Vernière ◽

...

Keyword(s):

Burkina Faso ◽

Sweet Orange ◽

Citrus Canker ◽

Negative Control ◽

Citrus Species ◽

Xanthomonas Citri ◽

First Report ◽

Mexican Lime ◽

Volkamer Lemon ◽

Wide Range

Citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of economic importance in tropical and sub-tropical citrus-producing areas (EPPO-PQR online database). X. citri pv. citri causes severe infection in a wide range of citrus species, and induces erumpent, callus-like lesions with water-soaked margins leading to premature fruit drop and twig dieback. It has consequently been subjected to eradication efforts and international regulations. It was first described on the African continent in South Africa at the beginning of the 20th century, from which it was eventually eradicated. Since 2006, several outbreaks caused by phylogenetically diverse strains of X. citri pv. citri have been reported from several African countries (Ethiopia, Mali, Senegal, and Somalia). In July 2011, citrus canker in Burkina Faso was suspected in the area adjacent to the Sikassso Province of Mali where X. citri pv. citri has been confirmed. In November and December 2012, leaves of clementine (Citrus clementina), lemon (C. limon), Volkamer lemon (C. volkameriana), sweet orange (C. sinensis), tangelo (C. paradisi× C. reticulata), and mandarin (C. reticulata) were collected from orchards with trees showing symptoms of citrus canker in the Comoé, Houet, and Kénédougou provinces of Burkina Faso. Isolations performed using KC semi-selective medium (4) recovered 45 Xanthomonas-like strains. All Xanthomonas-like strains were tentatively identified as X. citri pv. citri by PCR (4/7 primers) using IAPAR 306 and sterile distilled water as the positive and negative controls, respectively (3). Among these, two strains (LK4-4 and LK4-5) produced a ‘fuscans’-like brown diffusible pigment, a phenotype never reported previously for X. citri pv. citri. MultiLocus Sequence Analysis targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,2) fully identified seven strains from Burkina Faso (LJ301-1, LJ303-1, LK1-1, LK2-6, LK4-3, LK4-4, and LK4-5) as X. citri pv. citri (and not to any other Xanthomonas pathovars pathogenic to citrus or host range-restricted pathotypes of pathovar citri), and more specifically as sequence type ST2 which is composed mostly of pathotype A strains of X. citri pv. citri (2). The same seven strains were inoculated to at least four leaves of each of grapefruit cv. Henderson, Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58 (C. latifolia), and sweet orange cv. Washington Navel, using a detached leaf assay (2). All strains developed typical erumpent, callus-like tissue at wound sites on all citrus species inoculated. No lesions developed on the negative control (sterile 10 mM tris buffer). Koch's postulate was fulfilled after reisolation of Xanthomonas-like yellow colonies from symptoms on Mexican lime produced by the seven strains. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (3) and produced the expected 468-bp amplicon in contrast with the PCR negative control. To our knowledge, this is the first report of X. citri pv. citri in Burkina Faso. Citrus canker-free nurseries and grove sanitation should be implemented for reducing the prevalence of Asiatic canker in Burkina Faso and a thorough survey of citrus nurseries and groves in the region should be conducted. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) J. S. Hartung et al. Phytopathology 86:95, 1996. (4) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005.

Download Full-text

First Report of Xanthomonas citri subsp. citri causing Citrus Canker on lime in Rio Grande do Norte, Brazil

Plant Disease ◽

10.1094/pdis-11-20-2498-pdn ◽

2021 ◽

Author(s):

Lucas Amancio ◽

Ana Dulce Botelho Baia ◽

Elineide B. Souza ◽

Rui Sales Júnior ◽

Andreia Mitsa Paiva Negreiros ◽

...

Keyword(s):

Rio Grande ◽

Citrus Canker ◽

Negative Control ◽

Culture Collection ◽

Bacterial Suspension ◽

Citrus Species ◽

Bacterial Strains ◽

Xanthomonas Citri ◽

Tropical Regions ◽

First Report

Citrus canker caused by Xanthomonas citri subsp. citri is one of the most important citrus diseases in the world (Gottwald et al. 2002), mainly for citrus-producing countries with humid sub-tropical regions such as United States, Argentina, and Brazil, where losses may be significant (Behlau et al. 2020). In the state of Rio Grande do Norte (RN), Brazil, citrus production is expanding and shows social and economic importance for small farmers, which produced approximately 297 tons of lime in this state in 2019 (IBGE 2021). In December 2019, we observed symptoms of erumpent lesions with margins surrounded by yellow haloes on leaves and fruit of the lime (Citrus aurantifolia cv. ‘Galego’) (about 5% incidence) in a plantation located in the municipality of Mossoró, RN (05°12'21.1"S, 37°19'16"W). Samples were collected from the lime orchard, and five bacterial strains (CCRMXC01 to CCRMXC05) showing yellow, convex, mucoid colonies were isolated in a nutrient-yeast-dextrose-agar medium (NYDA). Pathogenicity tests were performed on sweet orange (C. sinensis cv. ‘Pêra’) and lime (C. latifolia cv. ‘Tahiti’) seedlings. Four wounds per leaf (upper side) were carried out with an entomological pin and 10 µl of a bacterial suspension (108 CFU mL-1) were deposited on each wound. The negative control consisted of leaves treated with sterile distilled water (SDW). For each citrus species, we used four replicates per strain and one leaf with four wounds per replicate. Inoculated leaves developed erumpent lesions with margins surrounded by yellow haloes six days after inoculation (DAI) in both citrus species, while leaves treated with SDW remained symptomless. Nine DAI, we reisolated the pathogen and performed rep-PCR (REP, ERIC, and BOX-PCR) analyses (Gama et al. 2018) with the strains inoculated and reisolated to confirm the identity of the strains and to fulfill Koch’s postulates. The strains were stored at the Culture Collection Rosa Mariano (CCRM) of the Phytobacteriology Laboratory at the Universidade Federal Rural de Pernambuco. The five strains reisolated showed the same REP, ERIC, and BOX-PCR profiles as the strains used for inoculations. The molecular identification was performed sequencing the dnaK, fyuA, gyrB, and rpoD genes (Young et al. 2008). Each fragment was sequenced in both the forward and reverse directions. Using the BLASTn tool, we observed that sequences of the dnaK (GenBank MW218913 to MW218917), fyuA (GenBank MW218918 to MW218922), and rpoD (GenBank MW218928 to MW218932) genes of the strains CCRMXC01 to CCRMXC05 showed 100% of identity with the sequences of these genes from the type strain (ICMP 24T) and of other strains of X. citri subsp. citri (ICMP 21 and ICMP 7493), while sequences of gryB (GenBank MW218923 to MW218927) of the former strains showed 100% identity with the gyrB sequence of the strains ICMP 24T and ICMP 7493 and 99,85% identity with strain ICMP 21. This short variation in the sequence of the gyrB gene also may be observed among strains of X. citri subsp. citri available in NCBI database (https://www.ncbi.nlm.nih.gov/). The phylogenetic analysis performed using Bayesian inference and the concatenated sequence of all the type or representative strains of species and pathovars of Xanthomonas available in GenBank showed that the strains CCRMXC01 to CCRMXC05 clustered together with strain ICMP 24T with 1.0 posterior probability. To our information, this is the first report of X. citri subsp. citri causing citrus canker on lime in RN state, Brazil.

Download Full-text

Resolving the structure of phage-bacteria interactions in the context of natural diversity

10.1101/2021.06.27.449121 ◽

2021 ◽

Author(s):

Kathryn M Kauffman ◽

William K Chang ◽

Julia M Brown ◽

Fatima Aysha Hussain ◽

Joy Y Yang ◽

...

Keyword(s):

Microbial Communities ◽

Host Range ◽

Large Scale ◽

Genomic Diversity ◽

Interaction Networks ◽

Bacterial Strains ◽

Quantitative Estimates ◽

Phage Evolution ◽

Marine Vibrio ◽

In The Wild

Microbial communities are shaped by viral predators. Yet, resolving which viruses (phages) and bacteria are interacting is a major challenge in the context of natural levels of microbial diversity. Thus, fundamental features of how phage-bacteria interactions are structured and evolve in "the wild" remain poorly resolved. Here we use large-scale isolation of environmental marine Vibrio bacteria and their phages to obtain quantitative estimates of strain-level phage predator loads, and use all-by-all host range assays to discover how phage and host genomic diversity shape interactions. We show that killing in environmental interaction networks is sparse - with phage predator loads low for most bacterial strains and phages host-strain-specific in their killing. Paradoxically, we also find that although overlap in killing is generally rare between phages, recombination is common. Together, these results indicate that the number of hosts that phages infect is often larger than the number that they kill and suggest that recombination during cryptic co-infections is an important mode of phage evolution in microbial communities. In the development of phages for bioengineering and therapeutics it will be important to consider that nucleic acids of introduced phages may spread into local phage populations through recombination, and that the likelihood of transfer is not predictable based on killing host range.

Download Full-text

First Report of Xanthomonas citri pv. citri-A Causing Asiatic Citrus Canker in Mayotte

Plant Disease ◽

10.1094/pdis-01-13-0128-pdn ◽

2013 ◽

Vol 97 (7) ◽

pp. 989-989

Author(s):

J. Hoarau ◽

C. Boyer ◽

K. Vital ◽

T. Chesneau ◽

C. Vernière ◽

...

Keyword(s):

Indian Ocean ◽

Sweet Orange ◽

Citrus Canker ◽

Negative Control ◽

Sterile Water ◽

Citrus Species ◽

Indian Ocean Region ◽

Southwest Indian Ocean ◽

Mexican Lime ◽

Ocean Region

Asiatic citrus canker, caused by Xanthomonas citri pv. citri, is a bacterial disease of major economic importance in tropical and subtropical citrus-producing areas. X. citri pv. citri pathotype A can cause severe infection in a wide range of citrus species and induces erumpent, callus-like lesions with water-soaked margins evolving to corky cankers and leading to premature fruit and leaf drop and twig dieback on susceptible/very susceptible cultivars. A chlorotic halo is typically visible around canker lesions on leaves and young fruit, but not on mature fruit and twigs. This quarantine organism can strongly impact both national and international citrus markets. Long distance dispersal is mainly through infected propagative material. Asiatic citrus canker occurs on most islands in the Southwest Indian Ocean region (Comoros, Mauritius, Reunion, Rodrigues, and Seychelles islands), but was not yet reported in Mayotte (EPPO-PQR available at http://www.eppo.int ). In May 2012, typical canker-like symptoms were observed on sweet orange (Citrus sinensis) groves on Mtsamboro islet and soon after on the main island of Mayotte, mostly on sweet oranges, but also on Tahiti limes (C. latifolia) and mandarins (C. reticulata). Eighty-one Xanthomonas-like strains were isolated using KC semi-selective medium (4) from disease samples collected from both commercial groves and nurseries on different Citrus species located all over the island. Sixteen Xanthomonas-like isolates were tentatively identified as X. citri pv. citri based on a specific PCR assay with 4/7 primers (3). All strains but the negative control, sterile water, produced an amplicon of the expected size similar to X. citri pv. citri strain IAPAR 306 used as positive control. Multilocus sequence analysis targeting six housekeeping genes (atpD, dnaK, efp, gltA, gyrB, and lepA) (1,2) fully identified three strains from Mayotte (LJ225-3, LJ228-1, and LJ229-11) as X. citri pv. citri (and not other xanthomonad pathovars pathogenic to citrus or host range-restricted pathotypes of pathovar citri), and more specifically as sequence type ST2 composed of pathotype A strains of X. citri pv. citri (2) (including all strains from the Southwest Indian Ocean region). Eight strains were inoculated by a detached leaf assay (2) to Mexican lime SRA 140 (C. aurantifolia), Tahiti lime SRA 58, sweet orange cv. Washington Navel, alemow SRA 779 (C. macrophylla), and tangor cv. Ortanique (C. reticulata × C. sinensis) and developed typical erumpent, callus-like tissue at wound sites for all Citrus species, fulfilling Koch's postulates. Xanthomonas-like yellow colonies were reisolated from symptoms produced by the eight strains inoculated on Mexican lime. Boiled bacterial suspensions were assayed by PCR with 4/7 primers (3) and produced the expected 468-bp amplicon in contrast with the negative control (sterile water). No lesions developed on the negative control consisting of inoculations by 10 mM tris buffer (pH 7.2). Citrus canker-free nurseries and grove sanitation should be implemented for decreasing the prevalence of Asiatic canker in this island territory. References: (1) N. F. Almeida et al. Phytopathology 100:208, 2010. (2) L. Bui Thi Ngoc et al. Int. J. Syst. Evol. Microbiol. 60:515, 2010. (3) J. S. Hartung et al. Phytopathology 86:95, 1996. (4) O. Pruvost et al. J. Appl. Microbiol. 99:803, 2005.

Download Full-text