| Blackleg(phoma stem canker)is an important fungal disease on Brassica napus(canola,oilseed rape)and many other cruciferous vegetable crops worldwide,it can cause severe economic losses to the industry of oilseed rape.This disease is caused by two closely related fungal species in the genus Leptosphaeria,one of which is Leptosphaeria maculans and the other is L.biglobosa.Relatively,L.maculans is generally considered to be the major contributor to yield losses in oilseed rape.Blackleg was reported to occur in China in the early 2000 s,and the casal agent is L.biglobosa.However,the epidemiological features of blackleg have been poorly elucidated in this country.Meanwhile,the question remains as to whether or not L.maculans exists in China.In this study,large-scale surveys on occurrence of blackleg of oilseed rape were carried out to clarify the epidemiology of blackleg in China.Moreover,factors affecting the epidemics of blackleg(climate,oilseed rape type,cultivation pattern and weevil activity)were analyzed from the survey data.In addition,a novel mycovirus LbBV1 was identified in L.biglobosa,and its biological characteristics and cross-species transmission were studied to provide clues of using this mycovirus to control blackleg of oilseed rape and other fungal diseases.Main results are summarized as follows:1.Occurrence and distribution status of blackleg of oilseed rape in China was clarified.During 2016-2022,extensive field surveys on blackleg of oilseed rape were done in oilseed rape planting areas in China,covering 2250 fields in 19 provinces,municipalities and autonomous regions.The results showed that blackleg existed in all the 19 surveyed provinces,and the overall percentages of oilseed rape fields with blackleg was 62%.The frequently-occurring provinces are Gansu,Qinghai,Shaanxi and Hubei,the average percentages of oilseed rape fields with blackleg is 77%,the values of the highest blackleg incidence ranged from 78% to 100%,and blackleg occurrence indices(BOI)ranged from33 to 46.In contrast,Chongqing,Shanghai and Yunnan had a rare occurrence of blackleg,the percentages of oilseed rape fields with blackleg were lower than 30%,the values of the highest blackleg incidence were lower than 12%,and the BOI valus were lower than 8.The results of fixed-point surveyed showed that blackleg mainly occurred at the ripening stage of oilseed rape.It occurred on leaves,stems,pods and seeds of oilseed rape,thereby causing phoma leaf spots,phoma stem canker,phoma pod rot and phoma seed rot,respectively.Among these symptom,phoma stem canker was the major symptom,it occurred both at the stem base(12% to 59%)and on upper stems(41% to 88%).Most phoma stem canker(70%)belonged to epidermal cankers,while some phoma stem cankers(30%)could reach to stem pith.2.Factors affecting epidemics of blackleg in China were elucidated.Blackleg in winter oilseed rape started at the bolting stage(phoma leaf spot),and peaked at the flowering and ripening stages(phoma stem canker,phoma pod rot,phoma seed rot).Phoma stem canker was the most common symptom,which was mostly associated with wounds or cracks on stems of oilseed rape.In winter oilseed rape planting areas,stem cracks are usually caused by low temperature,the rates of this type samples ranged from 73% to 88%.In spring oilseed rape planting areas,weevil-associated phoma stem canker was usually associated with weevil activities(wounds caused in laying eggs on the stem and feeding on the pith of the stems,the holes formed after the mature larvae escaping from the stems),the rates of this type ranged from 26% to 69%.Climate conditions affected the prevalence of blackleg,and there are differences in influencing factors between winter and spring oilseed rape planting areas.Temperature and rainfall from the sowing stage to the seedling stage(October to December)of oilseed rape are the main factors for blackleg epidemics on winter oilseed rape.In the whole period of winter oilseed rape,the monthly average temperature ranged from 6℃ to 22℃,the monthly cumulative rainfall ranged from 30 mm to 139 mm.The results of correlation analysis showed that the average temperature in October,November and March was negatively correlated with blackleg occurrence index(BOI),the monthly cumulative rainfall in October was positively correlated with BOI(P < 0.05).Rainfall was the main factor for blackleg epidemics on spring oilseed rape planting areas.In the whole period of spring oilseed rape,the monthly average temperature ranged from8℃ to 20℃,the monthly cumulative rainfall ranged from 25 mm to 107 mm.The results of correlation analysis showed that the monthly cumulative rainfall from April to September(except July)were positively correlated with BOI(P < 0.05).Blackleg incidence of a total of 1,297 oilseed rape fields in winter oilseed rape was analyzed and the results showed that oilseed rapes cultivated under the continuous dryland-cropping pattern were more prone to blackleg than those cultivated under the paddy land/dryland-cropping pattern.The average BOI under the dry land/dry land-cropping pattern fields was 39,which was significantly higher than the average BOI of 27 under the paddy land/dry land-cropping pattern fields(P < 0.01).Blackleg incidence of a total of 1,979 oilseed rape fields was analyzed and the results showed that oilseed rapes of Brassica campestris and B.juncea were more susceptible than that of B.napus to blackleg.The average BOI of B.napus was25,which was significantly lower than B.juncea(53 BOI,P < 0.001)and B.campestries(40 BOI,P < 0.05).Blackleg also occurred on other cruciferous vegetables crops besides oilseed rape.3544 samples collected from different host crops in China were isolated and identified in this paper,the results showed that the 2534 strains belonged to L.biglobosa.3.Low temperature and rainfall were found to be conductive to sexual reproduction by L.biglobosa on diseased stubbles of oilseed rape.Dynamic monitoring of the life cycle of L.biglobosa was observed for four consecutive seasons from 2017 to2021.The results revealed that the sexual development of L.biglobosa in winter oilseed rape planting areas(such as Wuhan,Hubei province)started from October and lasted up to the end of April of the next year,which covered the whole oilseed rape growth period.Development of pseudothecia and the release of ascospores were greatly affected by temperature and rainfall,the monthly average temperature was negatively correlated with the proportion of mature pseudocyst in the range of 5℃ to 20℃(r = ﹣0.55,P < 0.05),and the proportion of monthly rainfall days was positively correlated with the proportion of mature pseudocyst and the density of ascospores(r = 0.50 and r = 0.70,respectively,P <0.05).4.Limited role of conidia of L.biglobosa as the primary infection source was observed.Inoculation of conidia of L.biglobosa on the cotyledons of oilseed rape showed that hyphae formed after conidial germination,and then they invaded tissues of oilseed rape through stomata and wounds.The hyphae of L.biglobosa exsited in the intercellular space,where they grew transversely and longitudinally.Inoculation of conidia of L.biglobosa on the true leaf petioles of oilseed rape showed that L.biglobosa could spread longitudinally downwards along the petioles after colonization,but could not spread to stems.Inoculation of conidia of L.biglobosa on root of oilseed rape showed that the conidia could infect the radicles and grew endophytically.Hyphae spread upward from the root tissues to the stem tissues through the intercellular space with the growth and development of oilseed rape.However,the biomass of L.biglobosa in oilseed rape plants dropped sharply at the flowering stage,and formation of phoma stem canker was not observed on the stems.Therefore,conidia of L.biglobosa might play a limited role as the primary infection source of blackleg.5.A novel mycovirus(LbBV1)was identified in L.biglobosa,it has no visible effects on growth,development and pathogenicity of L.biglobosa,however,it caused pathogenicity debilitation when it was transmitted to Botrytis cinerea.Leptosphaeria biglobosa botybirnavirus 1(LbBV1)was characterized in L.biglobosa strain GZJS-19.It did not cause obvious symptoms in L.biglobosa,and had no significant effect on pathogenicity of L.biglobosa either.However,cross-species transmission of LbBV1 from L.biglobosa to B.cinerea resulted in hypovirulence of the recipient B.cinerea strain t-459-V.The cross-species transmission was artificially realized through mixed incubation of the conidia of L.biglobosa and B.cinerea on agar medium(e.g.,PDA)and on stems of oilseed rape with the transmission efficiency of 4.6% and 18.8%,respectively.Niche overlapping of L.biglobosa and B.cinerea exists in nature,RNA sequencing was carried out on L.biglobosa and B.cinerea isolates obtained from Brassica samples co-infected by these two pathogens.The results showed that at least two mycoviruses were detected in both fungal groups.These results indicate that cross-species transmission of mycoviruses may occur frequently in nature and result in the phenotypical changes of newly invaded phytopathogenic fungi.These results contributed to further understand the epidemics of blackleg in China,they will be helpful for designing relevant prevention and control policies and measures against blackleg of oilseed rape.Meanwhile,the finding about the cross-species transmission of LbBV1 provided a new perspective for using LbBV1 as a biological control agent. |
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