Identification And Biological Characteristics Of Root Rot Pathogen On Blueberry

The root rot of blueberry is one of the most important diseases in blueberry production.It not only effects the quality and yield,but also causes the whole plant death and serious economic losses.With the development of blueberry planting areas,different planting environment,variety distribution and seedling origin have also resulted in the diversity of diseases.Two different types of root rot were found on blueberry production of root diseases in some areas in 2013-2017.One was caused by Fusarium,the other was caused by Calonectria.Both two kinds of pathogen could damage roots of blueberry,and gradually made leaves and branches wilt,and eventually caused plant death.In order to identified the pathogen clearly,and provide theoretical basis for management of this disease,pathogenic fungi isolated from the roots of blueberry were identified based on morphological characteristics and molecular techniques.The differences between isolates in the pathogenicity and the biological characteristics were also study.The PCR detection of root rot pathogens was conducted in this paper.1.4 species of root rot pathogens on blueberry were obtained by isolation and identification4 representitive isolates of root rot pathogens on blueberry were obtained by isolation and identification: The result showed that the isolate HNCS02 Bb was identified as F.equiseti and HNCS02 Da was identified as F.commune.The isolate SDRN02 Ba was identified as Fusarium oxysporum.YNQJ01 Aa clustered together with Calonectria ilicicola reference strain.The morphological characteristics of the root pathogens were observed.The Fusarium isolates showes diversity of morphological characteristics,and all produced typical macroconidia of Fusarium.3 isolates were chosen with differentmorphological characteristics: HNCS02 Bb aerial mycelium was strong,woolly,the surface of colony was white to pink;the colony of HNCS02 Da was white to purple on the surface and purple on the back;the colony of SDRS02 Ba was white to purple,and the back was purple.The colony of isolates YNQJ01Aa’s which belongs to the genus Calonectria is white to yellow on the surface on PDA,and white to light yellow on the back.According to the morphological characteristics of conidia,it is identified as the pathogen of the genus Calonectria.Molecular identification: 3 isolates of Fusarium and 1 isolate of Calonectria were chosen for further molecular identification and phylogenetic analysis.The ITS gene of all 4 isolates showed the same result as the morphologicai identification.The phylogenetic trees of Calonectria isolates were combined EF-1 alpha gene,beta-tubulin gene and Histone 3 gene.Fusarium phylogenetic trees were constructed by the EF-1 alpha gene.The result showed that the isolate HNCS02 Bb was identified as F.equiseti(F.incarnatum-equiseti complex)and HNCS02 Da was identified as F.commune.The isolate SDRN02 Ba was identified as Fusarium oxysporum of Fusarium oxysporum complex.YNQJ01 Aa clustered together with Calonectria ilicicola(anamorph Cylindrocladium parasiticum)reference strain.At the same time,we also found a stem disease caused by the genus Calonectria.The isolate was identified as C.canadensis(anamorph C.canadense).The strain of this disease was also used in PCR test in this study.2.There were differences on biological characteristics among 4 species of blueberry root rot pathogensThe biological characteristics of 4 representative strains of root root showed that:there was no significant difference in colony diameter on different carbon sources of F.equiseti strain HNCS02 Bb,the strain of F.commune HNCS02 Da,and F.oxysporum strain SDRS02 Ba.The optimum carbon of C.ilicicola strain YNQJ01 Aa was soluble starch.The optimum nitrogen of HNCS02 Da was cysteine;the optimum nitrogensources of HNCS02 Bb were cysteine and Na NO3;the optimum nitrogen of SDRS02 Ba and C.ilicicola strain YNQJ01 Aa was Na NO3.The strain could grow well on the culture medium of PDA,OA and blueberry media.HNCS02 Bb,HNCS02Da could grow in the range of 10℃-35℃,and SDRS02 Ba and YNQJ01 Aa could grow in the range of 10 ℃-30 ℃.The optimum growth temperature of HNCS02 Da and SDRS02 Ba was 25 ℃-30 ℃,and the optimum temperature of YNQJ01 Aa and HNCS02 Bb was 25℃.The growth of all strains in 3 different light conditions showed no obvious difference.3.There were differences on sensibility of different cultivars to 4 species of blueberry root rot pathogensThe 6 cultivars were inoculated by root-irrigation method and the differences of sensibility was observed.The cultivar ’O’ neal’ was chosen for the inoculation of the leaves and stems to compare the difference of pathogenicity among the tested strains.The results showed that there were significant differences among 4 strains.The strain HNCS02 Bb had no pathogenicity to ’Elliot’and ’Sharp blue’ after 30 days of innoculation,and HNCS02 Da had pathogenicity to all the tested cultivars,but the symptoms were different when different cultivars were inoculated 30 d.The strain SDRS02 Ba had no pathogenicity to ’Elliot’,and YNQJ01 Aa had no pathogenicity to the ’Sharp blue’.The strain YNQJ01 Aa not only infected the roots of blueberry,but also infected leaves and branches,and the extension was rapid.HNCS02 Bb and HNCS02 Da infected blueberry leaves and branches besides the roots of blueberry,but the pathogenicity was weak,while SDRS02 Ba had no ability to infect the leaves but also infected the roots and branches.4.The species-associated PCR primers of C.ilicicola and F.equiseti were designed and reported primers of F.oxysporum and F.commune were determinedAmong the 4 species of root pathogens identified in this study,there were plentyprevious studies by others about F.oxysporum and F.commune PCR detection,therefore,the reported primers can be used for detection of two species.The study on C.ilicicola and F.equiseti is rare.In this paper,specific primers are designed and verified for these two species.The C.illicola species specific primer Cail4F/R was based on beta-tubulin gene,the length of the product fragment is 362 bp,and F.equiseti specific primer Feq EF/R was based on the EF-1 alpha gene,and the length of the product is 200 bp.The specificity of the two pairs of primers was proved,only the target DNA was amplified,but the species of the non-target blueberry root rot fungus,the pathogen of the other blueberry and the DNA of blueberry didn’t not show positive result.The sensitivity of primers was also tested.Cail4F/R could detect the target fungus DNA of 100 pg in 25 μl PCR system,and Feq EF/R could detect the target fungus DNA of 1 ng in the 25 μl PCR system.For the specific detection of F.oxysporum and F.commune,primers designed by predecessors were used in this study,and the sensitivity and specificity of primers C1 /C2 and FO1/FO2 were determined.The combination of 4 primers can achieve the detection of pathogens in blueberry root.The annealing temperature of all primers was similar,and can be amplified under the same reaction conditions.