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MORPHOLOGICAL, BIOCHEMICAL AND GENETIC CHARACTERIZATION OF CITRUS CANKER PATHOGEN (XANTHOMONASAXONOPODIS) FROM CITRUS CULTIVARS OF PUNJAB, PAKISTAN.

Byline: I. Shehzadi and S. Naz

Keywords: citrus canker, biochemical tests, Xanthomonas axonopodis, Biochemical assay, PCR.

INTRODUCTION

Citrus is an important fruit crop with annual worldwide production of 2.4 million metric tons in 2015-16. In Pakistan, citrus fruits are grown on an area of 206,569 hectares with annual production 2.36 million tons in 2015-16 and more than 98% of citrus fruit is produced only in Punjab (Pakistan Bureau of Statistics, 2016).

The yield and quality of citrus fruits faces many production constraints including diseases caused by fungal, viral, bacterial and nematode pathogens. Citrus nematode, citrus gummosis, citrus canker, citrus greening and citrus tristeza virus are commonly prevalent in Pakistan. Among these diseases, citrus canker affects the plant growth and fruit quality in all citrus growing areas of the world (Pruvost et al., 2002). The infectious agent of the disease is Xanthomonas axonopodis (Sahi et al., 2007). From Pakistan it was first reported in Punjab and after which it becomes epidemic throughout the country. All the cultivars are susceptible for citrus canker such as grapefruit, sweet oranges, lemons, limes, rootstocks and their hybrids (Gottwald et al.,2002).

Citrus canker has been grouped into three types A, B, C based on host rang and geographical distribution (Graham et al, 2004). The Asiatic form A caused by Xac is most virulent form throughout the world including Pakistan (Canteros et al., 2017). The citrus canker B (false canker) caused by X. citripv. Auranti folii was reported in Argentina, Uruguay and Paraguay on lemon (Citrus limon). The cancrosis C also caused by X. citripv. aurantifolii has been found only in Sao Paulo state in Brazil. The two known hosts of C type canker are mexican lime and sour orange (Schubert et al., 2001).

The symptoms caused by citrus canker are pustules to necrotic lesions consisting of erumpent corky tissues surrounded by oily or water-soaked margins and yellow halo. The disease severity causes defoliation, dieback, premature fruit drop and blemished fruit. Lesions on the fruit are most economically important damage, which makes the fruit not acceptable for fresh market, and have very little price. The integrated control measures of the disease are the production of disease free stock, copper sprays and decrease in pathogen spreading caused by leaf miner or wind (Das, 2003; Schubert and Sun, 2003; Graham et al., 2004).

Early rapid and specific detection of canker-associated bacterium is important for plant disease management (Mansfield et al., 2012). Without diagnosis of the disease, it cannot be control at specific time (Mccartney et al., 2003). Conventional assays to detect and identify bacterial pathogen involved isolating the pathogen on selective media and its identification through biochemical analysis (Gottwald et al., 2001). But molecular based methods can overcome many shortcomings in conventional methods. PCR based molecular markers are generally more specific and much reliable (Grote et al., 2002).

Keeping in view the reduction in market value of citrus due to citrus canker in Pakistan, the present study was aimed to provide knowledge about the prevalence of CBC in Pakistan citrus varieties. By the use of the biochemical assay and genetic diversity by PCR, 95 citrus samples were screened for Xac isolates. Genetic diversity by PCR assay targeting different genomic regions of the pathogen was first studied in Pakistan.

MATERIALS AND METHODS

Collection of samples: For the detection and assessment of incidence of CBC ninety-five different citrus samples were collected from "Citrus Research Institute (CRI), Sargodha, Pakistan" in summer (June, July, August) 2016. The symptoms were yellow to dark brown lesions as shown in Fig 1. The leaves were collected in labeled polythene bags and stored at 4AdegC prior to use.

Isolation of pathogen and preparation of Glycerol stocks: The canker lesion along with 2mm of the peel around was excised from infectious citrus leaves and left for 10 minutes in sterile water for the release of the bacteria. The extract of the tweezed bacteria was streaked on the selective media GYP (Galactose yeast peptone: peptone 0.5g, yeast extract 0.5g, agar-agar 1.5g, glucose 1g, distill water 100ml) media for 2 days on 28AdegC. Suspected single colonies of X. axonopodis were further purified and stored in 50% glycerol stocks at -80AdegC.

Morphological Characterization: The morphological characteristics such as colony appearance, shape, margins and pigmentation were used to identify Xac pathogen associated with citrus canker. The bacterium shape was identified by the standard gram's staining technique. The bacterium shape, size, arrangement and staining reaction was observed under 100X microscope.

Biochemical tests: The pathogen was identified biochemically by performing twenty different biochemical tests using API-E 20 strip. The strips were used by following the manual described by the bio Merieuxs API kit.

DNA Extraction: Two different methods were optimized for the DNA extraction. First method was used for the Xanthomonas cells grown in vitro, while the method II was used for the extraction of total DNA from the canker lesions on leaves from all citrus varieties. Each method is described below:

In first method total purified genomic bacterial DNA was isolated by phenol chloroform method from in vitro cultured bacteria Sambrook et al. (1989). In second method citrus leaves with canker lesions were cut and washed with 70% ethanol. The total genomic DNA was extracted by following CTAB (hexa decyl trimethyl ammonium bromide) method by Murrayand Thomson (1980) with some modification (Naz et al., 2015).

DNA quantification: The DNA quantification was done by measuring the optical density (OD) of DNA by UV/VIS Spectrophotometer (Optizen NANO Q).

PCR amplification: Samples were analyzed by conventional PCR Primus-96 Thermal cycler by three different primer sets that amplified 16S rDNA and ITS region of the citrus canker pathogen (Table 2). The Dream Taq green PCR master mix (2X) Cat#1018 was used which comprises of 2X Dream Taq Green buffer, 0.4 mM each dNTPs, 4 mM MgCl2. The final concentration of primer and template from cultured cells and plant material was 0.4uM and 15ng respectively. The PCR thermo cycling conditions were an initial denaturation steps at 94AdegC for 3 min followed by the 36 cycles of denaturation at 94AdegC for 30s, annealing at 50AdegC for 30s and elongation at 72AdegC for 45s, followed by extension at 72AdegC for 10 minutes. The amplified product was subjected to electrophoresis through 1% agarose gel and analyzed in gel documentation system (Wealtec, Dolphin)

Cloning and Sequence analysis: The amplified PCR products were purified using GF-1 Gel recovery kit (K# GF-GF-100), cloned into pTZ57R/T vector (Invitrogen) and transformed into E.coli competent cells. After cloning the plasmid was purified by GF-1Plasmid purification kit (K#GF-PL-100). Recombinant plasmid was confirmed by colony PCR and restriction digestion. The plasmid was sequenced by ABI Prism DNA sequencer (Perkin-Elmer, USA). The sequences were aligned and compared with reference sequences available on GenBank database by BLASTn (https://blast. ncbi. nlm. nih.gov/Blast.cgi.).

Phylogenetic analysis: The Phylogenetic analysis was done using the neighbor joining method in MEGA7 (Kumar et al., 2016). The reference sequence from GenBank and plasmid sequence was used for evolutionary analysis and Candidatus Liberibacter asiaticus sequence from GenBank (Accession No. KY038851.1) was used as out-group. The tree with bootstrap values was created from 500 replicates taken to represent the evolutionary history and branches reproduced in less than 50% bootstrap replicates were collapsed. The evolutionary distances were calculated by p-distance method and the analysis involved 7 nucleotide sequences. All positions containing gaps and missing data were eliminated. A total of 376 positions were included in the final dataset.

Table 1. Oligonucleotide primers pair used for Polymerase chain reaction (PCR).

Primers###Sequences###Product size(bp)###Amplified regions

HF###5'-CACGGTGCAAAAAATCT-3'###222

HR###5'-TGGTGTCGTCGCTTGTAT-3'###16S rDNA

XCF###5'-AGGCCGGTATGCGAAAGTCCCATCA-3'###424###ITS

XCR###5'-CAAGTTGCCTCGGAGCTATC-3'

XACF###5'-CGCAT CCCCACCACCACCACGAC-3'###500###ITS

XACR###5'-AACCGCTCAATGCCATCCACTTCA -3'

Table 2. Morphological Characteristics of Xanthomonas axonopodis.

Isolates###Pigmentation###Configuration###Shape###Texture###Margins###Opacity

Xac01###Yellow###Small, single###Rod###Mucoid###Smooth###Translucent

Xac02###Yellow###Small, single###Rod###Mucoid###Smooth###Semi-Translucent

Xac03###Yellow###Small, single###Rod###Mucoid###Smooth###Translucent

Xac04###Yellow###Small, single###Rod###Mucoid###Smooth###Semi Translucent

Xac05###Yellow###Small, single###Rod###Mucoid###Smooth###Translucent

Table 3. Biochemical test of Xanthomonas axonopodis pvcitri used for differentiating various trains.

Tests###Biochemical test###Results Coloration###Xac1###Xac2###Xac3###Xac4###Xac5

ONPG###Beta-galactosidase###Colorless###-###-###-###-###-

ADH###Arginine dihydrolase###Yellow###-###-###-###-###-

LDC###Lysine decarboxylase###Yellow###-###-###-###-###-

ODC###Ornithine decarboxylase###Yellow###-###-###-###-###-

CIT###Citrate utilization###Blue###+###+###+###+###+

H 2S###H2S production###Black deposit###+###+###+###+###+

URE###Urea hydrolysis###Yellow###-###-###-###+###-

TDA###Deaminase###Brown red###+###+###+###+###+

IND###Indole production###Red###-###-###-###-###-

VP###Acetoin production###Pink###+###+###+###+###+

GEL###Gelatinase###No diffusion of black###+###+###+###+###+

GLU###Glucose fermentation###Blue/blue-green###-###-###-###-###-

MAN###Mannitol fermentation###Blue/blue-green###-###-###-###-###-

INO###Inositol fermentaion###blue/blue-green###-###-###-###-###-

SOR###Sorbitol Fermentation###blue/blue-green###-###-###-###-###-

RHA###Rhaminose Fermentation###Yellow###+###+###+###+###+

SAC###Sucrose fermentation###Yellow###+###+###+###+###+

MEL###Melibiose fermentation###Blue###-###-###-###-###-

AMY###Amygdalin fermentation###Blue###-###-###-###-###-

ARA###Arabinose Fermentation###Yellow###+###+###+###+###+

OX###Oxidase###Violet###+###+###+###+###+

Table 4. Different varieties of citrus used to evaluate different set of primers, based on 16S (HF/HR), and internally transcribed spacer region (XACF/XACR, XCF/XCR) for PCR identification of Xac.

Groups###Varieties###Total.###HF/HR###XCF/XCR###XACF/XACR

###No. of###(%age mean of###(%age mean of###(% age mean of

###samples###identification)###identification)###identification)

Grapefruit###Shamber(SH)###5###94###95###100

###Star ruby(Kakalikova et al.)###5###60###60###70

###Red blush(RB)###5###75###80###80

###Pink Ruby(PR)###5###80###80###80

###Ray Ruby(RR)###5###60###60###58

###Ruby blood(Rb)###5###85###90###90

###Pomelo(P)###5###55###60###70

age of Susceptibility toxic###78###80###82

Kumqat###Nagami(N)###5###15###20###17

###Marumi(MA)###5###55###60###55

###Meiwa(ME)###5###30###40###40

% age of Susceptibility toxic###30###40###40

Lemons and lime###Volkameriana###5###50###60###60

###Eureka###5###60###60###60

###Mesero###5###35###40###35

###Lisbon###5###98###100###100

###Local mitha###5###78###80###80

###Pehawasrimittha###5###78###80###78

###Tahiti lime###5###80###80###75

###Kagzi lime###5###80###80###80

###Lakeland###5###98###100###98

###% age of Susceptibility to Xac

RESULTS AND DISCUSSION

The identification of Xac was carried out from citrus different cultivars by morphological, biochemical and molecular markers.

Morphological Characterization: The leaves lesions placed on GYP agar plate showed the bacterial colonies after 24 hours at 28AdegC identified the bacterium as Xac. The colony color of the isolates was variable from light yellow to dark yellow (Fig 2A). The colonies of Xac bacterial isolates were small, round, mucoid and yellow due to accumulation of xanthomandian. The results were in line with previous study of Das (2003) who reported that seven isolates of the Xac isolates were gram negative and yellow in color on selective medium. The typical yellow coloration of the colonies was due to the pigmentation of xanthomonadin, which is produced by different Xanthomonas. The colonies were mucoid due to the production of extracellular polysaccharide slime by the addition of glucose in the culture medium (Singh and Thind, 2014). The size, shape and arrangement of colonies were found to be small to medium, convex and single (Fig 2B). The characteristics of the bacterium are in table 2.

Gram staining test from 5 isolates was found as gram negative and rod shaped bacteria. All morphological properties were consistent with those pre-viously described for pathotype A by Sun et al. (2004).

Biochemical Characterization: The biochemical properties were studied by API-E20 strip, which involves different biochemical tests. In biochemical tests the bacterial isolates were positive for the citrate utilization, H2S production, urea hydrolysis, deaminase, acetoin production and hydrolysis of gelatin. But they were negative for the fermentation of sorbitol, mannitol and glucose. The different biochemical tests are listed in Table 3: Beta galactosidase, arginine hydrolase, lysine decarboxylase, ornithine decarboxylase test were all negative for all strains. Xac1, Xac2, Xac3, Xac4 and Xac5 all strains showed the positive results towards citrate utilization and H2S production. Xac 04 was positive for urea hydrolysis while all other strains were negative for the respective test.

Juhasz et al. (2013) also reported that Xac isolates were positive for catalase, KOH and H2S production; hydrolysis of starch, gelatin liquefaction and fermentation of different sugars such as sucrose. Deaminase utilization test for all the strains was positive with brownish red coloration. In present study the isolates fermented different carbohydrates such as rhaminose, sucrose, arabinose and oxidase, but they were negative for the fermentation of sorbitol, mannitol and glucose Glucose mannitol sorbitol and inositol fermentation tests were negative by Xac1, Xac2, Xac3, Xac4 and Xac5. Xac1, Xac02, Xac03, Xac04 and Xac05 were positive for rhaminose and sucrose utilization, while melibiose and amygdalin fermentation was negative. These findings are in line with Mohammadi, et al. (2001) who suggested that eighteen different isolates of Xac from Marathwada region of India showed utilization of arabinose, rhaminose and sucrose.

Detection of Xac by PCR: The PCR was used for the identification of the CBC causing pathogen by using three different primer sets amplifying different regions of the citrus canker pathogen. An amplified fragment of 222bp from 16S rDNA, 424bp from XCF/XCR and 500bp from XACF/XACR was observed from all collected citrus cultivars. Results of PCR amplification have been depicted in Table 4.Specific PCR products were detected from all primer sets. The18-mer oligonucleotide HR/HF primer pair tested for Xac was amplified by 78% Xanthomonas species in grapefruit group of citrus. The highest degree of disease 95% was identified by shamber and lowest 60% by pomelo in grapefruit. The predicted amplified product was obtained from 30% isolates of Xac from different varieties of citrus belonging to kumquats group and 70% from lemon and lime group members.

The 16S rRNA sequences based primers (HF/HR) amplified specific region in all citrus cultivars, these results were corroborate with those reported by Cubero and Graham (2002).

Zafarullah et al. (2016) reported that the 16S rDNA gene was considered unsuitable due to the high levels of sequence similarity in this region and revealed very limited diversity. Therefore ITS based primers have high discriminating power, so specific primers based on ITS region amplified a specific band in all strains. Two sets of primer based on ITS region XCF/XCR and XACF/XACR were used for the rapid identification of all strains. The XCF/XCR primer amplified a PCR product of Xac by 80% varieties of citrus belonging to grapefruit group. The 40% isolates from kumqats, 70% from lemon and lime were identified by this primer pair and no amplified product was observed from healthy plants. The other set of primer XACF/XACR amplified PCR product of 500bp from 82% grapefruits, 40% from kumquats and 77% from isolates belonging to lemon and lime group(Fig 4).

In these results all the citrus cultivars amplified by XACF/XACR primer pair as Park et al. (2006) also mentioned that this primer set amplified DNA fragment from Xac in different citrus cultivars.

In next step the rate of disease susceptibility among different citrus groups was calculated by percentage mean. The highest percentage of disease susceptibility was observed in grapefruit while kumqats showed the lowest percentage of disease (Table 4).To check the specificity of the primer other negative controls such as healthy plant and non-template control showed negative result for the pathogen. These findings were similar with the previous reported findings by Burhan et al.(2007) that intensity of disease severity was partially similar in different cultivars such as maximum (56.62%) was found in Mars Early, immediately followed by Olinda Valencia. Singh et al. (2014) also find out that grapefruit have the highest degree of disease susceptibility among different citrus groups due to high level of gene expression which code for extra cellular polysaccharide (EPS) in Xac pathogen to enhance the canker development.

Phylogenetic Tree: A phylogenetic tree constructed by neighbor joining method clustered all Xanthomonas axonopodis taxa into a monophyletic group. The group was divided into two subgroups. First group was consisting of the isolates with Accession No. CP011827.2AF442739.1, 20163533C_XCF, and other group containedDQ991181.1, KF926678.1 and AF442744.1 (Fig. 5). The sequence of ITS region of X. axonopodis isolates from shamber (shown as 3C_XCF) showed 100% homology to reference sequence of X. axonopodis pv. citri isolate from China (Accesion No. CP011827.2). Xacwas 99%similar to the sequence of the region of the isolate of USA (AF442739.1). The other group showed less similarity with the sequence of the ITS region of the pathogen. The Candidatus Liberibacter asiaticus (Accession No.KY038851.1) appeared as a separate group in evolutionary analysis which has no similarity with the other groups. Overall results confirmed that infectious pathogen A type is prevalent in Pakistan.

The sequencing and phylogenetic analysis showed 100% homology to pathotype A strain from USA and China which confirmed the prevalence A type CBC in selected regions of citrus germplasm of Pakistan.

Citrus canker is a devastating disease, which is affecting the economy of Pakistan very badly. In present study we have detected the pathogen by morphological, biochemical and molecular characterization. Pathogen was found to be affecting almost all the citrus varieties to different extent. Therefore there is a need to follow up the correct cultivation of citrus cultivars to decrease disease incidence in nurseries and to prevent the spreading pathogen (Xac) to new emerging groves. This study would be helpful for future planning of characterization and control of this disease.

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