Studies On Epidemics Of Bacterial Fruit Blotch On Grafted Watermelon Seedlings And Biological Control Of BFB

Bacterial fruit blotch(BFB)of cucurbits is a devastating disease with a worldwide distribution.It is caused by Acidovorax citrulli(Ac).To date,BFB-tolerant/resistant commercial cultivars of watermelon as well as many other cucurbits are not available.As grafted watermelon seedlings are high resisitant with soil borne diseases and stress environment,the production of grafted watermelon seedlings developed rapidly.Many commercial grafted-seedling nurseries were set up in China,which brought great economic benefits in many regions of China.However,as the high temperature,high humidity and high density of cultivation of watermelon seedlings always occur in grafting nurseries,and many workers were involved in the grafting procedure,BFB has broken out recently in many grafting nurseries,thereby threatening production of grafted watermelon seedlings in China.The study investigated the effects of seed-and spray-inoculation of different inoculum loads of A.citrulli on bottle gourd rootstocks and watermelon scions on BFB epidemics in grafted watermelon seedlings.Efficacy of culture filtrates of A.niger Y-1 in suppression of BFB was investigated.Meanwhile,we isolated an Ac-infecting phage and investigated its basic biology and taxonomic status.Main results are summarized below.1.Relationship between BFB epidemics and environmental factor(temperature,humidity)was clarified,relationship between BFB and seed inoculum loads of Ac was also investigated.Results showed that the seeds of bottle gourd rootstocks could lead to BFB on seedlings under the temperature ranging from 20°C to 32°C,and relative humidity higher than 60%.The disease incidence and disease index of the treated seedlings were increased with the increase of temperature and humidity.When infected seedlings were cultivated under the same environment,the higher the inoculum was loaded in seeds,the higher disease incidence and disease index on the seedlings.Infected seeds resulted in BFB on seedlings even at the inoculum load of 10~2 cfu/seed.When infected seeds grew to seedlings,the BFB symptoms was first displayed on the cotyledons,cells of A.citrulli were located in the vascular tissue and palisade tissue of cotyledons.A.citrulli infected hypocotyl via cotyledons under the favor environmental conditions.A.citrulli could colonize at the xylem and phloem of vascular tissue of the seedling hypocotyl.Finally,the seedlings became collapsed and death with severe BFB symptoms.2.The effects of infected rootstocks and scions on BFB of grafted watermelon seedlings were elucidated.The primary infection was estabolished by seed inoculation with Ac and the secondary infection was established by spray inoculation with Ac.The results showed that BFB developed rapidly when grafted on Ac-infected bottle gourd rootstocks with different seed inoculum loads of Ac.The Ac-infected rootstocks resulted death of the grafted watermelon seedlings at 6 days post grafting(dpg),even at the seed inoculum load is 103 cfu/seed or disease aeras on cotyledons were less than 5%.When infected watermelon scion seedlings were available for grafting,the scions displayed BFB symptoms caused grafted seedlings dead.On the other hand,some scions without BFB lesions before grafting and developed from inoculated seeds also exhibited BFB symptoms at 6 dpg.The values of disease incidence for the grafted watermelon seedlings were statistically different among the three seed inoculum loads.On the secondary spread of BFB,the survival rates,disease incidences and disease index were significantly different between inoculum loads of A.citrulli,even the amount of A.citrulli in the tissues of rootstock or scion reached only at 10~2 cfu/g.The number of grafted seedlings with graft union infection is significantly(P <0.05)higher than that of cotyledon infection,although the grafted union was healed on some grafted seedlings.The BFB symptoms were observed on the cotyledons,it is suggest these grafted seedlings could be the potential source of BFB secondary infection in the field.3.Effect and mechanism of the culture filtrates of A.niger Y-1 against A.citrulli were elucidated.The p H values were decreased with the inoculation time increase.The HPLC analysis showed that the compounds of the organic acids in the culture filtrates of A.niger Y-1 were oxalic acid(OA)and citric acid(CA),and mainly the oxalic acid.In vitro assays,inhibition zones against A.citrulli were formed by the six-day-old culture filtrates(CF)of A.niger and OA(60 mmol/L)with the diameter of 15 mm,which are significantly larger than that of citric acid.However,the CF,CA and OA inhibited growth of A.citrulli on King’s B agar medium and their inhibitory effect disappeared when their p H values was adjusted to 7.0.It is suggested that the antibacterial activity depending on the acidic environment.Germination of watermelon and bottle gourd seeds treated with CF or OA could be inhibited,after seeds washing will relieve the inhibition.The potting experiment showed that CF applied on bottle gourd and watermelon seeds with 30 min effectively suppressed severity of seedling blight,disease incidences of seedlings were significantly higher than that of A.citrulli seed treatment(P <0.05),and the efficacy was comparable to the seed treatment with HCl(1%,v/v).The results suggest that CF of A.niger has potential for control of BFB.4.A novel phage(Ac P1)infecting Acidovorax citrulli was isolated from soil and its basic features(host range and response to environment)were characterized.Ac P1 is a lysogenic phage.Its survival temperature is below 35℃.The range of survival p H values for Ac P1 is between 4 to 13.Ac P1 is sensitive to UV and irradiationand chloroform.Ac P1 were obvsersed filamentous by TEM obversion.Ac P1 has a high host-specificity to A.citrulli,only group II of A.citrulli could be infected and lysogenic,according to this characteristic,Ac P1 has the potential ability to rapidly detect different groups of A.citrulli.