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experiments


INTRODUCTION:
Chilli along tomato, potato and eggplant is member of solanaceae family.Chilli crop originated in central and South America and later European spice dealers introduced it to rest of the world (Perry et al., 2007).Since medieval times, chilli is known for its nutritional, medicinal and cuisine value.Capsaicin is the major bioactive substance while chilli also contains carbohydrates, fats, and proteins (Salam, 2015;Kantar et al., 2016).In Pakistan, chilli is grown on area of 31 thousand acres with production of 82 thousand tons.Sindh province contributes 85% of the national production and Kunri chilli market of Sindh is considered as the biggest chilli market in Asia (Mangan and Ruthbah, 2018).Chilli crop is prone to both abiotic and biotic stresses.Among biotic stresses, bacterial diseases of chilli have worldwide distribution which cause minor losses to complete crop failure.The major bacterial diseases of chilli include wilt (Ralstonia solanacaearum), bacterial speck (Pseudomonas syringae) and bacterial leaf spot (Xanthomonas campestris pv vesicatoria) (Oerke, 2006;Shin and Yun, 2010).Bacterial diseases are reported from different chilli growing areas of Pakistan and responsible for significant crop losses (Begum et al., 2012).In chilli like any other crop, different management strategies are followed to counter diseases which include quarantine, crop rotation, physical, chemical, and biological control (Abbasi et al., 2002;Algam et al., 2010;Le et al., 2020;Dowarah et al., 2021).However, evaluation of innate immunity in germplasm is considered as most robust, economical, long lasting, eco-friendly control measure against plant diseases.The natural ability of X. campestris pv.vesicatoria to cause the significant losses in chilli crop is dependent upon virulence factors (Kim et al., 2008).The distribution of virulence factors in pathogenic population not only varies but continuously changing due to mutations, genetic recombination, gene and genotype flow etc.Additionally, abiotic factors such as circadian clock, temperature, humidity play their part (Ahmad and Ahmad, 2022).Similarly, varietal resistance against local population also varies and needed to be assessed regularly against prevailing pathogenic population for sustainable crop production.Subsequently, screened R-plant and virulent pathogen could be used for future germplasm screening programs.In spite of the fact that chilli crop is regularly attacked by bacteria in Pakistan but the information regarding indigenous resistant source against prevailing bacterial ingresses is hardly available.Therefore, this study was planned to evaluate the inherent immunity of chilli germplasm against field and seed borne bacterial pathogens.

OBJECTIVES:
The research was conducted with following objectives (1) characterization of putative pathogenic bacterial isolates from chilli based upon biochemical traits.(2) unraveling of innate resistance source in chilli germplasm against local putative bacterial isolates.MATERIALS AND METHODS: Collection and isolation of putative bacterial isolates from planting material: Symptomatic chilli plant samples were collected from Mianwali, Taxila, Chakwal, Islamabad and Rawalpindi during 2021-22.Additionally, seeds of chilli germplasm (Apollo, Golden Hot, California Wonder, ATX Green, Hot Pepper, Skyline) were also investigated for the presence of bacterial pathogens (Schaad et al., 2001).Forty surface sterilized seeds of each cultivar (10seeds/plate) were subjected to count infection percentage.The seed infection percentage was calculated by formula (Boukaew et al., 2017).Seed Infection Percentage = Number of infected seeds X 100 Total number of seeds Phenotypic and biochemical characterization: Colonies of putative bacterial isolates were observed for colony colour and shape on nutrient agar (NA) and on yeast extract-dextrose-Calcium Carbonate (YDC) media.Different biochemical tests such as KOH test (Suslow et al., 1982), catalase test (Schaad et al., 2001), oxidase, urease production (Goszczynska et al., 2000), sulphide or motility test were performed by using sulfide indole motility (SIM media).Oxygen requirement of putative isolates were also assessed (Akbar et al., 2015).Pathogenicity assays: Detached asymptomatic or healthy leaves of chilli cultivars (Apollo, Skyline, Hot pepper, Gola Peshawari) and for seedling inoculation (TAX, Sky Star, Golden hot) methods were used to study inbred resistance and susceptibility against X. vesicatoria and P. syringae isolates.Fresh cultures of X. vesicatoria and P. syringae were prepared in sterilized distilled water.The OD 600 nm was adjusted at 0.4 (10 5 cfu/ml).Asymptomatic or healthy leaves of chilli were collected and placed on moist sterilized filter paper in petri plates.Leaves were pin pricked and subsequently inoculated separately with X. vesicatoria and P. syringae (Sahin and Miller, 1998).Sterilized water treated leaves were considered as negative control.Leaves were regularly monitored for 7 days post inoculation (dpi) for the appearance of disease symptoms.Three chilli cultivars (Tax, Sky star and Golden Hot) were evaluated against X. vesicatoria and P. syringae by foliar spray method while plants treated with sterilized distilled water were considered as mock treatment (Aslam et al., 2017).Inoculated chilli plants were kept in green house and regularly observed for 7 dpi for the appearance of symptoms.Disease severity was assessed by using Vallejos et al. (2010) rating scale; 1 = no disease symptoms, 2 = slight to moderate yellowing and slight necrosis, 3 = extensive yellowing and moderate necrosis, and 4 = complete necrosis.Invitro sensitivity assays: Sensitivity of X. vesicatoria and P. syringae against antibiotics such as penicillin (10 ug), streptomycin (10ug), tetracycline (30 ug), erythromycin (15ug) was assessed by plate poison technique (Bauer et al., 1966).Commercially available antibiotic (Oxoid®) discs were used.Fresh bacterial suspensions were swabbed on NA plates.Four discs were placed on plates and www.sciplatform.com

Research Manuscript
zone of inhibition around individual disc was measured.Plates swabbed with bacterial suspension but without discs were used as a control.Four replicates of each antibiotic were measured.Well diffusion method (Valgas et al., 2007) was used to evaluate different concentrations (50ppm, 100ppm 200ppm and 1000ppm) of copper sulphate against putative bacterial strains.Plates were placed in incubator at 28°C for 24 hours.RESULTS: Isolation of putative bacteria from chilli: In total, 6 putative bacterial isolates were isolated.Five putative isolates (GP, TAX, AAR, CM, ISL) were collected from chilli infected leaf segments while 1 putative isolate (APO) from seeds of different cultivars (figure 1).Out of 6 chilli cultivars investigated for seed borne infection, only two cultivars (Skyline, Hot pepper) carried no seed infection while the maximum seed infection was found in Apollo and ATX green (20%).Characterization based on biochemical traits: All the putative bacterial isolates were grown on NA and YDC culture media.Putative isolates collected from leaves appeared yellow on YDC media while bacteria isolated from seeds made cream-colored colonies.All six isolates (GP, TAX, AAR, CM, ISL, APO) formed loop and found strictly aerobic and catalase positive.However, putative isolates were unable to produce hydrogen sulphide, oxidase and arginine dihydrolase (figure 2, table 1).Screening of chilli germplasm: Detached leaf method depicted that cultivars (Apollo, Skyline, Hot pepper) showed susceptibility against APO (Pseudomonas syringae) while no symptoms were appeared on Gola Peshawari.It is pertinent to mention that APO isolate was initially isolated from chilli cultivar Apollo.Three cultivars (Gola Peshawari, Skyline and Apollo) showed susceptibility while Hot pepper showed resistance against GP (Xanthomonas vesicatoria) isolate in comparison to water treated leaves.In seed inoculation method, chilli cultivars (TAX, Sky Star and Golden Hot) tested against APO and GP isolates.Our data showed that TAX cultivar of chilli was resistant while Sky star and Golden hot were moderately resistant against GP.Sky star was found resistant while TAX and Golden hot were found susceptible against APO after 10 dpi (figure 3).Pakistan is also at cross road of world economies due to China Pakistan economic corridor (CPEC) which increase chances of introduction of invasive pathogen species.Likewise, chilli crop in Pakistan is also under stress due to bacterial diseases which already blighted the future of growers, traders and exporters.The current study was planned to evaluate the potential of chilli germplasm against prevailing seedborne and foliar diseases of bacterial origin.Pathogen virulence analysis and subsequent host resistance are two key areas which needed a consistent surveillance and evaluation.In current study, chilli seed infection up to 20% was found with Pseudomonas syringae.Seed is always considered as major vehicle of pathogen dispersal in new geographical areas where it previously not found.Studies showed that even a small friction of seed infection could lead to crop epidemics (Shenge et al., 2010;Umesha, 2020).Therefore, Pseudomonas syringae presence at such a high percentage is looming threat to sustainable chilli production in Pakistan.It is imperative to mention that Xanthomonas vesicatoria was also isolated from symptomatic leaf segments collected from different geographical areas.In Pakistan, chilli is grown in warm areas which increases its vulnerability against Xanthomonas vesicatoria.Xanthomonas vesicatoria is pathogen who has the potential to cause losses of great magnitude in solanaceous crops.Recently, Ahmad and Ahmad (2022) (Costa et al., 2021;Utami et al., 2022).Detached leaf and seedling inoculation assays showed varietal variation when it comes to resistance against Pseudomonas syringae and Xanthomonas vesicatoria.Licciardello et al. (2022) also determined the susceptibility of citrus against Xanthomonas citri pv.citri and X. citri pv.aurantifolii by detached leaf assays.Our results showed that there is variation among plant cultivars to pathogen due to presence of different set of resistant genes to fend off hemibiotroph (P.syringae) and biotroph (X.vesicatoria).Copper based assays showed bacterial resistance which also reported by Basim et al. (2005) while working on Taiwanese isolates of Xanthomonas campestris pv.vesicatoria.However, bacteria showed susceptibility against different antibiotics (erythromycin.penicillin, streptomycin, tetracycline).McManus and Stockwell (2000) discussed the success story of streptomycin and oxytetracycline in control phytobacteria especially Erwinia amylovora causal organism apple and pear blight.It is imperative to mention that regulators such as environment protection agency (EPA) also allowed both streptomycin and oxytetracycline for the control of phytobacteria.Contrarily, antibiotics are not promoted for the control of bacterial diseases in country like Pakistan CONCLUSION: Our preliminary study revealed the prevalence of Xanthomonas vesicatoria and Pseudomonas syringae both in seed and field plantations of chilli.However, chilli germplasm showed variations when it comes to resistance to both bacteria.It is worth to mention that local bacterial isolates were sensitive to antibiotics but resistant to copper sulphate.Therefore, it is recommended that chilli germplasm not only regularly evaluated against prevailing pathogens but use of antibiotics must be promoted in future disease control program.

CONFLICT OF INTEREST:
Authors have no conflict of interest.

Figure 1 :
Figure 1: Bacterial growth (a) and sympatomatic chilli leaves (b).Characterization based on biochemical traits: All the putative bacterial isolates were grown on NA and YDC culture media.Putative isolates collected from leaves appeared yellow on YDC media while bacteria isolated from seeds made cream-colored colonies.All six isolates (GP, TAX, AAR, CM, ISL, APO) formed loop and found strictly aerobic and catalase positive.However, putative isolates were unable to produce hydrogen sulphide, oxidase and arginine dihydrolase (figure2, table1).

Figure 2 :
Figure 2: Biochemical assays of bacterial isolates a) Gram negative bacteria forming the loop when treated with 3% KOH.b) Catalase test indicating bubbles production on treatment with 10% H2O2 c) Yellow bacterial growth on YDC medium.d) Creamy bacterial growth on YDC medium.Screening of chilli germplasm: Detached leaf method depicted that cultivars (Apollo, Skyline, Hot pepper) showed susceptibility against APO (Pseudomonas syringae) while no symptoms were appeared on Gola Peshawari.It is pertinent to mention that APO isolate was initially isolated from chilli cultivar Apollo.Three cultivars (Gola Peshawari, Skyline and Apollo) showed susceptibility while Hot pepper showed resistance against GP (Xanthomonas vesicatoria) isolate in comparison to water treated leaves.In seed inoculation method, chilli cultivars (TAX, Sky Star and Golden Hot) tested against APO and GP isolates.Our data showed that TAX cultivar of chilli was resistant while Sky star and Golden hot were moderately resistant against GP.Sky star was found resistant while TAX and Golden hot were found susceptible against APO after 10 dpi (figure3).

Figure 3 :
Figure 3: Statistical measurement of mean disease rating scale against X. vesicatoria and P. syringae.Invitro sensitivity assays: All four antibiotics (erythromycin.penicillin, streptomycin, tetracycline) formed zone of inhibition in plates containing Xanthomonas vesicatoria and Pseudomonas syringae by poison plate method after 24 hours. .Maximum zone of inhibition against GP (22.5) and APO (22.5) was formed by tetracycline and streptomycin respectively (figure 4, 5).

Figure 5 :
Figure5: Zone of inhibition shows the efficacy of antibiotics against Xanthomonas vesicatoria.E; erythromycin, P; penicillin, S; streptomycin, T; Tetracycline.Despite, bacterial isolates tested against different concentrations of copper sulphates but no zone of inhibition was observed on media plates (figure6).

Figure 6 :
Figure 6: Invitro evaluations of bacterial strains A) APO and B) GP against copper sulphate (50ppm,100ppm,1000 ppm).DISCUSSION: Pakistan is ranked 7 th most vulnerable country to climate change which causes many abiotic and biotic stresses.Pakistan is also at cross road of world economies due to China Pakistan economic corridor (CPEC) which increase chances of introduction of invasive pathogen species.Likewise, chilli crop in Pakistan is also under stress due to bacterial diseases which already blighted the future of growers, traders and exporters.The current study was planned to evaluate the potential of chilli germplasm against prevailing seedborne and foliar diseases of

Table 1 :
Characterization of bacterial isolates based on biochemical assays.
reported high disease incidence and severity in tomato crop infected by bacterial leaf spot caused by Xanthomonas vesicatoria in different districts of KPK province of Pakistan.Studies showed that the pathogen under favourable conditions causes losses up to 50 % in different parts of Pakistan.It is worth to mention that European Cooperation in Science and Technology (COST) has actually run a program EuroXanth COST Action (2017-21) to counter the threat posed by members of family Xanthomonadacea which includes genus Xanthomonas and Xylella to food security