01-NafisaSudan-Oxolinicacid-700-706

See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/301753261 OXOLINIC ACID RESISTANCE IN FIELD ISOLATES OF XANTHOMONAS CAMPESTRIS PV Experiment Findings · August 2008 DOI: 10.13140/RG.2.1.2273.8807 CITATIONS READS 22 5 authors, including: Sami Adam DAL Group 6 PUBLICATIONS 22 CITATIONS SEE PROFILE All content following this page was uploaded by Sami Adam on 01 May 2016 The user has requested enhancement of the downloaded file Page No 700-706 LSIJ, Vol 2, No.3, July 2008 OXOLINIC ACID RESISTANCE IN FIELD ISOLATES OF XANTHOMONAS CAMPESTRIS PV MALVACEARUM By NAFISA E AHMED, SAMI M ADAM, *MOHAMED O IDRIS, KHALID D ABUIDRIS AND YOUSIF S ADAM Agricultural Research Corporation, Wad Medani, Sudan *University of Khartoum, Faculty of Agriculture, Khartoum, Sudan ABSTRACT A total of 51 isolates of Xanthomonas campestris pv malvacearum were isolated from infected cotton leaves collected from Gezira Research Farm (GRF) and three sites in the Gezira Scheme All samples were from the cotton variety Barakat 90 From these, 28 isolates were selected for detailed characterization In season 2003/04, two strains of the bacterium resistant to oxolinic acid (OA) were isolated from a cotton field at GRF that had received OA treatment In 2004/05, four OA resistant strains were detected in a field at GRF that had received OA three times in the period from 1997-2005 Oxolinic acid resistant strains were detected at GRF from large water soaked angular leaf spots (LWALS) in season 2003/04 and from main vein blight (MVB) and stem lesions (SL) in 2004/05 The minimum inhibitory concentration of streptomycin to OA highly resistant strains at GRF had changed from 50µg/ml in 2003/04 to 250µg/ml in season 2004/05 Strains with resistance to streptomycin and ciprofloxacin were not detected in any of the tested isolates KEYWORDS: Cotton, bacterial blight, seed dressing, fungicides, bacterium, Gezira, Sudan INTRODUCTION Bacterial blight of cotton caused by Xanthomonas campestris pv malvacearum (XCM) is potentially the most damaging disease to cotton in the Sudan The disease became the most prevalent among commercial varieties and across the irrigated area It is responsible for heavy leaf shedding and fruit abscission and is considered a major cause of lower yields It can reduce seed cotton yield by 14-21% depending on the prevailing environmental conditions (Mohamed et al 2003) Under irrigated cotton production, seed dressing with fungicides/bactericides is being considered as a major component of the integrated disease management package Few fungicides/bactericides were recommended for use to control primary disease infection Mercuric fungicides were widely used across the irrigated schemes until its production was band and the bactericide copper oxide was released to replace it In 1997, resistance to copper oxide was reported in Gezira scheme and the product was withdrawn from use in Sudan in season 1997/98 Similar results were obtained in USA where copper resistance in pear orchard was reported and the product was withdrawn from use (Looper et al 1991) Oxolinic acid was introduced and released for large scale use in the irrigated schemes as seed treatment to control seed borne bacteria and protect the plants against secondary infection at the seedling stage Oxolinic acid is a quinolone antibiotic which is not widely used in agriculture for its indispensable importance as second line drugs for humans and animals Quinolones are synthetic antibiotics that act by inhibiting DNA synthesis in bacteria (Drlica and Zhao, 1997) 700 Ahmed, et al (2008) Oxolinic acid resistance in cotton in Sudan The original quinolones (oxolinic acid, nalidixic acid) have modest activity against gram negative bacteria only and documented to cause quick mutation in this group (Hikchi and Shimizu 1989; Hikichi et al 1998 and 2001; Nakamara et al 1989) Flouroquinolones were developed to extend the spectrum of quinolones to include gram +bacteria (Pseudomonas aerugensis) and gram –ve bacteria resistant to quinolones (Hirai et al 1986; Nakamara et al 1989) However, cross resistance could develop specially under inadequate dosing in very high population (Hikichi et al 2001) In Sudan, a variety of quinolones are currently in use for human treatment These include nalidixic acid for chronic urinary tract infections, ciprofloxacin for typhoid treatment and norofloxacin for other inflammatory diseases In agriculture, oxolinic acid is reported to have been used for control of bacterial diseases on vegetables such as cabbage, potato and onion in Japan in 1989 (Hikichi and Shimizu 1989) Recently, Oxolinic acid is used as a seed treatment against seedling rot and as a spray to control grain rot in rice caused by Burkholderia glumae Hikichi et al (1998) found that OAresistant B glumae isolates have a concomitant cross resistance to other quinolones In 2004, Maeda et al 2004 reported that field isolates from rice seedlings grown from oxolinic acid treated seeds in Japan were divided into sensitive, moderately resistant and highly resistant isolates In addition, they provided evidence that mutation is involved in the oxolinic acid resistance of field isolates The bactericide was evaluated for its efficacy to control fire blight disease of pome fruits orchards in Israel in 1997-2000 (Manulis et al 2004) In 1999, resistance was detected and the product was completely withdrawn from use in 2001 (Manulis et al 2003) It is well documented that use of oxolinic acid will inevitably lead to development of resistant forms of bacteria in the ecosystem (Shtienberg et al 2001) In season 2001/02 we observed that the efficacy of oxolinic acid to control cotton bacterial blight at the seedling stage in Gezira scheme had deteriorated and a number of unusual disease symptoms appeared In this study we investigated the resistance of Xanthomonas campestris pv malvacearum, the causal organism of bacterial blight of cotton, to oxolinic acid MATERIALS AND METHODS Isolation of bacteria Samples of infected leaves were collected from a cotton field treated with oxolinic acid in season 2003/2004 and from different cotton fields at GRF in season 2004/05 including a field that have received three OA treatments between 1997 and 2004 More samples were collected in season 2004/05 from locations in the Gezira Scheme, namely, Gamousy (G), Wadi Shaeer (W) and Elshamaly (Sh) All infected plant tissues were sampled from the cotton G barbadense variety barakat 90 referred to in the text as barakat In season 2003/04 Xanthomonas campestris pv malvacearum was isolated from stem lesions (SL) and from leaves showing extended vein blight (EVB), angular normal leaf spots (ANLS), symptoms produced normally by the new race (post-barakat), the only causal organism of cotton bacterial blight Similarly, the bacteria were isolated from angular water soaked leaf spots ( AWLS), large angular water soaked leaf spots (LAWLS) and main vein blight (MVB), symptoms known to be produced only by the old race (pre- barakat) to which all cotton varieties grown in Sudan are immune (fig 1) An isolate of Xanthomonas campestris pv malvacearum from old infected cotton leaves showing small necrotic angular spots collected in previous seasons from plants that did not receive OA, pressed and kept at the pathology laboratory, was used for comparison and referred to as new race original (NRO) Isolates collected in 2004/05 from the Gezira scheme and GRF were sorted out according to leaf symptoms into: restricted vein blight (RVB), Extended vein blight (EVB), normal angular leaf spots (NLS), large angular leaf spots (LLS), minute leaf spot (MLS) and circular leaf spot (CLS) (Fig 1) Pathogenicity test 48 hr cultures of the seven bacterial isolates from GRF in season 2003/04 were diluted in sterile distilled water to give approximately 106cfu/ml 701 Ahmed, et al (2008) Oxolinic acid resistance in cotton in Sudan The suspensions were used to inoculate the first four true leaves of weeks old cotton plants ． ． ．． ． ． ．． of the highly susceptible variety barakat 90 In season 2004/05 the isolates of X.c pv ．． ． ．．．．．．．． malvacearum were grouped into clusters according to their genetic diversity revealed by the PCR analysis (Adam et al 2008) Inoculated plants were kept in humid chamber to ensure high relative humidity for 10 days The experiments were established in randomized complete block design Five plants replicated times were used for each treatment Similarly treated cotton plants were kept un inoculated for comparison Inoculated plants were observed daily and disease reaction was recorded up to weeks after inoculation Determination of the minimum inhibitory concentration (MIC) of oxolinic acid The bacteria were cultured and maintained on nutrient agar (NA) medium In- vitro toxicity of different concentrations of oxolinic acid (Starner 20%), 50- 1000 µg/ml, on the different isolates of the bacterium was evaluated on the basis of bacterial growth inhibition MIC was determined using the disc diffusion technique Saturated discs (1cm diameter) with the bactericide suspension were placed on solid NA plates streaked with 48 hr cultures of the bacterium Discs saturated with sterile distilled water were included as checks Sensitivity of the same isolates to streptomycin sulfate and ciprofloxacin was examined for comparison Inhibition of bacterial growth was evaluated by measuring inhibition zone diameter The lowest concentration that did not show any visible growth inhibition by macroscopic evaluation was considered the MIC Each assay was repeated twice RESULTS AND DISCUSSION Pathogencity test All tested isolates produced characteristic symptoms of bacterial blight on inoculated cotton plants 10-15 days after inoculation These symptoms differ in expression and severity Minimum inhibitory concentration (MIC) of oxolinic acid It is evident from the inhibitory results in Table that the bacteria isolated from leaves showing normal angular necrotic spots collected from oxolinic acid treated plot in season 2003/04 were resistant to all tested concentrations These isolates represent the new race (post- barakat) referred to as ANLS which was previously found to be sensitive to oxolinic acid at 50µg/ml (Ahmed et al 2004) This rate is extremely higher than the rate recommended for other Gram negative bacteria (Manulis et al 2003) However, the bacteria isolated from plants showing main vein blight (MVB) were sensitive to oxolinic acid and inhibited by concentration as low as 50µg/ml (table 1) These symptoms are similar to those usually caused by the old race (pre- barakat) and were never reported before in any of the cotton varieties having B2B6 gene combination, the genes that conferred bacterial blight resistance in Sudanese cotton varieties The new race isolate (NRO) that was not exposed to oxolinic acid was inhibited by all concentrations tested (table 1) The isolates from leaves showing angular water soaked leaf spots (AWLS), large angular water soaked leaf spots (LAWLS) and diffused vein blight (EVB) were sensitive to oxolinic acid at 1000 and 500 µg/ml and were less sensitive to 250µg/ml where irregular slight inhibition occurred The MIC for these isolates is expected between 250 and 100µg/ml Isolates from stem lesions are also sensitive to oxolinic acid and their MIC is lower than 50µg/ml The isolates from large water soaked spots and water soaked main vein blight were very aggressive and caused symptoms that had never been reported before in barakat or any other cotton variety having B2B6 gene combination These symptoms are confined to a field at GRF that had been sown with barakat treated by oxolinic acid during the seasons 1997-2005 The disease intensity in this field was very high (93.8%) compared to 40, 38.5 and 31.2% in other fields growing the same variety but were not exposed to oxolinic acid treatment In this field it was observed that the symptoms included small and large necrotic and water soaked leaf spots, restricted and extended or diffused vein blight, stem lesions and circular leaf spots a very unusual symptoms of bacterial blight However, in other fields at GRF, growing the same variety but was not treated with oxolinic acid, only normal small angular leaf spots and restricted vein blight were reported This indicates that the repetitive use of oxolinic acid resulted in establishment of resistant strains of X.c pv malvacearum in the ecosystem 702 Ahmed, et al (2008) Oxolinic acid resistance in cotton in Sudan It is also evident from these results that the MIC for streptomycin sulfate was far less than 50µg/ml for all isolates except for the new race that was resistant to oxolinic acid where it was between 250 and 100µg/ml However, this isolate was never exposed to streptomycin sulfate treatment and this antibiotic was never used for agricultural purposes This suggests that this isolate can acquire resistance to streptomycin (table 1) Table indicates that all isolates collected from GRF and Gezira scheme in season 2004/05 were highly sensitive to streptomycin and ciprofloxacin However, there are variations among and within the strains isolated from similar symptoms at different locations in their reaction to oxolinic acid According to sensitivity of the isolates presented in table 2, there are at least three strains resistant to oxolinic acid at each tested location In total at Gezira Scheme there are groups representing 14 OA-resistant isolates These could be categorized according to our results into sensitive (MIC 50µg/ml), moderately resistant (MIC100 µg/ml), resistant (250 µg/ml) and highly resistant (MIC 500µg/ml) At GRF we detected two groups of isolates, one is highly resistant (MIC > 1000µg/ml) while the other coincide with the moderately resistant isolates at the Gezira Scheme ((MIC100 µg/ml) Accordingly, occurrence of at least four distinct groups of isolates that have a high degree of coincidence with the symptoms they cause and their oxolinic acid resistance is apparent Previous studies in 1996-1999, field isolates of X c pv malvacearum collected from different locations at Gezira and Rahad Schemes showed neither pathological difference (Ahmed et al 1997) nor genetic variability within the post- barakat race using PCR technique (Ahmed 2000) It is only in season 2002/03 we observed unusual symptoms of bacterial blight on cotton in the Gezira after the large scale use of oxolinic acid seed treatment in about 50 thousands hectare of cotton The disease was not controlled by oxolinic acid in season 2002/03 at some locations of Gezira scheme where the soil inoculums of the bacteria were historically known to be very high compared to the bactericide rate used Manulis et al (2003) demonstrated that the use of at- risk compounds where large bacterial populations exist should be avoided Moreover, oxolinic acid application to high populations of bacteria might result in the development of oxolinic acid resistant bacteria Oxolinic acid is the only antibiotic commercially used in agriculture and in the largest scale worldwide against bacterial blight of cotton in Sudan The bactericide was introduced in limited areas in other countries and withdrawn for development of resistance (Loper et al 1991) In Japan resistant strains of Burkholderia glumae pathogenic to rice to oxolinic acid was reported (Hikichi et al, 1998, Maeda et al, 2004) Maeda et al (2004) divided B glumae into groups according to their resistance to oxolinic acid which ranged from sensitive to highly resistant (higher than 100µg/ml) We detected four OA- resistant groups of X.c pv malvacearum that are resistant to 1000, 500, 250 and 100 µg/ml The results of the present study suggest that high resistance to oxolinic acid might develop among sensitive, resistant and moderately resistant strains, thus infecting the cotton crop, by multiple oxolinic acid applications In conclusion we observed that oxolinic acid resistance developed gradually during several seasons at GRF and development of more aggressive disease symptoms is evident in locations where oxolinic acid was used for several seasons Occurrence of more than one strain of the bacteria X.c pv.malvacearum is apparent in Gezira Research Farm and Gezira scheme Continuous use of oxolinic acid to control bacterial blight is expected to broaden diversity of strains and emergence of very aggressive strains that will threaten cotton production and breeding program of resistant varieties to the disease Therefore it is recommended that use of oxolinic acid in areas of high bacteria population should be avoided to maintain resistant strains to this product at very low level and to prevent distribution of this resistance across cotton production areas Cotton growers where resistance to oxolinic acid was detected are advised to conduct strict sanitation and to burn all plant debris immediately after harvest to reduce the amount of resistant inoculum In addition, Isolation of X.c.pv malvacearum strains from infected plant tissues at different locations and characterization of their response to oxolinic acid should be an integral part of bacterial blight management strategy in Sudan 703 Ahmed, et al (2008) Oxolinic acid resistance in cotton in Sudan Table 1: Inhibition of Xanthomonas campestris pv malvacearum growth (cm) by oxolinic acid and streptomycin sulfate (isolates from GRF) season 2003/04 Bacterial Oxolinic acid conc µg/ml Streptomycin conc µg/ml isolates 1000 500 250 100 50 1000 500 250 100 50 ANLS 0 0 0.5 0.3 0.1 0 MVB 0.7 0.7 0.5 0.3 0.1 0.5 0.5 0.4 0.3 0.3 NRO 0.5 0.5 0.3 0.3 0.2 1.5 0.8 0.6 0.5 0.5 LAWLS 0.7 0.4 * 0 0.8 0.6 0.4 0.3 0.1 AWLS 0.6 0.6 * 0 0.9 0.7 0.7 0.4 0.2 EVB 0.8 0.6 * 0 0.7 07 0.5 0.3 0.3 SL 0.9 0.8 0.8 0.6 0.5 0.8 0.6 0.5 0.4 0.4 control 0 0 0 0 0 *Slight irregular inhibition zone < mm diameter Table 2: Effects of oxolinic acid (µg/ml) in-vitro on growth of isolates of X.c.pv malvacearum from different locations at Gezira Scheme and GRF season 2004/05 Bacterial Oxolinic acid µg/ml Streptomycin sulfate Ciprofloxacin µg/ml isolates µg/ml 1000 500 1000 500 1000 500 250 100 250 100 250 100 RVB Sh 3.0 2.7 1.8 0.0 5.0 3.0 3.0 2.5 7.0 5.0 4.5 5.0 EVB Sh 6.2 3.5 2.9 2.1 5.5 3.6 3.3 2.9 6.3 4.8 5.1 4.8 NLS Sh 3.8 2.2 0.0 0.0 4.9 3.5 2.7 3.0 5.8 3.9 4.2 3.6 LLS Sh 2.6 0.0 0.0 0.0 5.6 3.9 3.1 2.8 5.4 3.7 3.3 4.4 RVB GRF 2.8 2.4 2.0 0.0 5.4 3.8 3.2 3.0 7.2 4.8 4.8 5.2 EVB GRF 3.5 3.2 2.7 2.0 6.3 4.6 4.8 3.6 6.7 6.0 5.3 4.9 NLS GRF 4.0 2.8 0.0 0.0 5.0 3.8 3.3 3.6 6.0 4.7 4.4 3.8 LLS GRF 3.8 3.0 1.2 0.0 5.8 4.8 4.0 3.4 5.6 4.3 4.0 4.4 MLS GRF 3.8 3.0 2.4 1.4 6.0 4.9 4.6 3.4 6.4 4.5 4.2 4.0 CLS GRF 4.2 3.1 2.7 0.0 5.6 5.0 4.2 4.0 6.8 5.2 4.4 4.6 RVB Gam 3.4 2.2 1.0 0.0 4.6 2.6 2.8 2.0 6.8 5.0 4.6 3.4 NLS Gam 3.5 2.0 1.0 0.0 4.6 3.5 2.8 2.0 6.5 5.9 4.8 4.0 MLS Gam 3.0 2.4 0.0 0.0 5.4 3.7 2.6 2.2 6.0 3.8 4.0 3.0 RVB W.Sh 5.1 3.8 1.7 1.2 4.9 4.1 4.4 3.6 6.5 5.9 4.3 4.0 EVB W.Sh 4.0 3.2 1.1 0.0 5.1 4.3 4.0 3.3 5.5 5.3 4.6 3.8 NLS W.Sh 3.6 3.0 1.4 0.0 4.8 4.0 4.2 3.3 5.6 5.2 4.7 3.8 LLS W.Sh 4.4 2.1 0.0 0.0 5.2 4.7 4.2 3.5 6.1 5.6 5.0 4.8 MLS W.Sh 4.5 3.1 2.8 1.4 5.3 4.4 3.7 3.0 6.2 5.5 5.1 4.9 Sh = Shamaly, GRF = Gezira Research Farm, Gam = Gamousy, W.Sh = Wadi shaeer 704 Ahmed, et al (2008) Oxolinic acid resistance in cotton in Sudan A B C D E F G H I J Fig 1: Cotton leaves and stems showing different symptoms of bacterial blight disease: A: Circular leaf spot D: Restricted vein blight G: Normal angular leaf spot J: minute leaf spot B: Extended vein blight C: Main vein blight E: Water soaked angular leaf spot F: Large leaf spot H: Large water soaked angular leaf spot I: Stem lesion 705 Ahmed, et al (2008) Oxolinic acid resistance in cotton in Sudan REFERENCES Ahmed, N E., Abdalla, M A., Sami, M A., Khalid, A., Yousif, S A 2004 A report on new mutants of Xanthomonas campestris pv malvacearum resistant to oxolinic acid Proceedings of 70th-71ist Meetings of 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