Effect of Different Inoculation Methods on Pathogenicity of Fusarium proliferatum to Alfalfa

Yinghua LUO, Mengteng LI, Congyu LI, Chenghao JIN, Xiangping LIU

College of Animal Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China

Abstract [Objectives] To find out the suitable inoculation method for Fusarium proliferatum.[Methods] Longmu 801 and TG4 were inoculated with rice grain-based inoculum at the bud and seedling stages and with root irrigation at the seedling stage, respectively, and then, the plant height, aboveground biomass, underground biomass, incidence rate and disease index of the alfalfa were determined to conduct comprehensive resistance evaluation. [Results] There was obvious difference in disease resistance between the two alfalfa varieties. Among the three inoculation methods, the relative plant height, relative aboveground biomass and relative underground biomass of Longmu 801 differed insignificantly (P>0.05), all around 100. There were significant differences in TG4 among different inoculation methods (P<0.05). The incidence rates and disease indices of Longmu 801 and TG4 inoculated with rice grain-based inoculum at the bud stage were the highest, and their incidence rates and disease indices were 73.3% and 100%, and 40 and 60, respectively, significantly higher than those under the other two inoculation methods. The incidence rates and disease indices of Longmu 801 and TG4 inoculated with rice grain-based inoculum at the seedling stage were the lowest, and their incidence rates and disease indices were 0% and 13.30%, and 0 and 5.33, respectively, significantly lower than those under the other two inoculation methods. [Conclusions] The inoculation time and inoculation method of the pathogen both affect the inoculation effect. Among the three inoculation methods, the inoculation with rice grain-based inoculum at the bud stage is the most pathogenic to alfalfa seedlings, and can be popularized in the resistance evaluation of alfalfa to root diseases.

Key words Alfalfa, Root rot, Fusarium proliferatum, Inoculation method, Disease resistance

1 Introduction

Root rot in alfalfa (MedicagosatiuaL.) is a chronic and potentially global disease that mainly occurs in the rhizomes, and it occurs in most alfalfa growing areas[1]. Research scholars at home and abroad have different opinions on the pathogens causing root rot in alfalfa. The possible reason is that the sampling is performed in different regions and under different ecological conditions. Most scholars believe that the disease is related toFusarium[2-3]. Root rot is a soil-borne disease that occurs in the roots of alfalfa. The pathogenic mechanism of its pathogenic bacteria is complex and various[3]. Many research results show that multipleFusariumspecies can lead to the occurrence of root rot in alfalfa, either alone or in combination, and the dominantFusariumspecies reported in different countries and regions are not exactly the same[4-5]. There are mainly eight pathogenic species ofFusariumthat cause alfalfa root rot in China, includingFusariumoxysporum,Fusariumsolani,Fusariumacuminatum,Fusariumequiseti,Fusariumsemitectum,Fusariumavenaceum,Fusariumproliferatum, andFusariumtricinctum[6]. Among them,F.proliferatumwas first isolated and named by Nirenberg in 1976[7]. There are many reports onF.proliferatumabroad, while there are few reports on it in China[6-7].F.proliferatumgenerally exists in soil and animal and plant organisms, and it is extremely extensive in geographical distribution[7]. In this experiment, the resistance of Longmu 801 and TG4 inoculated with same-dosageF.proliferatumby different methods (inoculation with rice grain-based inoculum at the bud stage, inoculation with rice grain-based inoculum at the seedling stage, and inoculation by root irrigation at the seedling stage) was compared. After 30 d of inoculation, the five resistance evaluation indices, plant height, aboveground biomass, underground biomass, incidence rate and disease index, of the experimental alfalfa materials were measured, respectively, and the resistance of the experimental alfalfa materials was evaluated comprehensively, so as to provide a theoretical basis for the follow-up research and new control technology ofFusariumroot rot of alfalfa.

2 Materials

2.1 Test alfalfa and strainThe alfalfa varieties tested in this experiment were Longmu 801 and TG4, provided by the Chinese Academy of Agricultural Sciences. The test strain for this experiment wasF.proliferatum, provided by the Pratacultural Science Laboratory of the College of Animal Science and Technology of Heilongjiang Bayi Agricultural University.

2.2 Main instruments and equipmentThe main instruments and equipment used included single-sided purification workbench (SW-CJ-2FD, Shanghai Sujing Industrial Co., Ltd.), biochemical incubator (SHP-250, Shanghai Senxin Experimental Instrument Co., Ltd.), electric constant-temperature drying oven (DGG-9240, Shanghai Senxin Experimental Instrument Co., Ltd.). The above instruments and equipment were provided by the Pratacultural Science Laboratory of the College of Animal Science and Technology of Heilongjiang Bayi Agricultural University.

3 Methods

3.1 Preparation of rice grain-based inoculumF.proliferatumstored at 4 ℃ was taken out rejuvenated on the PDA plate. After constant-temperature cultivation at 25 ℃ for 7 d, the hyphae and conidia were scraped down using a scalpel under sterile conditions, placed in a triangular flask filled with sterile water, shaken thoroughly to separate the conidia from the hyphae, and filtered to obtain a conidial suspension with a concentration of 1×106conidia/mL. A certain amount (50 g) of rice grains were poured into a petri dish with a diameter of 15 cm, added with 150 mL of distilled water, autoclaved (121 ℃ for 30 min), cooled to room temperature, mixed with 20 mL of the conidial suspension evenly, and incubated at 25 ℃ for 4 d to make the dish filled with hyphae for future use[8-9].

3.2 Disinfection and nursery of alfalfa seedsThe alfalfa seeds tested were disinfected with 75% alcohol for 2 min, disinfected with 1% sodium hypochlorite solution for 5 min, rinsed with sterile water 5 times and placed in sterile petri dish for 2 d to promote germination. After the seeds germinated, the seedlings with uniform growth were selected and transplanted to sterilized pots (13 cm in height and 15 cm in caliber), 10 seedlings in each pot. The seedlings were covered with 5-cm-deep sterile soil, and cultivated in an artificial climate room (temperature of 25 ℃, relative humidity of 80%)[10-12].

3.3 Experimental design

3.3.1Inoculation with rice grain-based inoculum at bud stage. The rice grain-based inoculum was inoculated to the surface of the soil (10 rice grains/plant) around the alfalfa seedlings (germinating for 2 d), and then covered with about 5-cm-deep sterile soil. Alfalfa seedlings inoculated with the same amount of sterile rice grains were taken as the control. Total four replicates were arranged for each variety. After inoculation, enough water was poured, followed by the cultivation in an artificial climate room (temperature of 25 ℃ and relative humidity of 80%). After 60 d of inoculation, when the alfalfa seedlings to be tested grew to about five-leaf stage, the disease in the roots was classified, the incidence rate and disease index were calculated, and the plant height, aboveground biomass and underground biomass were determined[13].

3.3.2Inoculation with rice grain-based inoculum at seedling stage. When the tested alfalfa seedlings grew for 30 d (around five-leaf stage), theF.proliferatum-infected rice grains were evenly spread around the rhizome of the alfalfa plants 1 cm under the surface, 10 rice grains/plant, and then covered with 5-cm-deep sterile soil. Alfalfa seedlings inoculated with the same amount of sterile rice grains were taken as the control. There were four replicates for each treatment. After 30 d of the inoculation, the disease was classified, the incidence rate and disease index were calculated, and the plant height, aboveground biomass and underground biomass were determined[13-14].

3.3.3Inoculation by root irrigation at seedling stage. A certain volume (1 mL) of the conical suspension of 1×106conidia/mL was irrigated around the roots of each of the 30-d-old (around five-leaf stage) tested alfalfa seedlings, respectively. Alfalfa seedlings irrigated with sterile water were taken as the control. Total four replicates were arranged for each treatment. After 30 d of the inoculation, the disease in the roots was classified, the incidence rate and disease index were calculated, and the plant height, aboveground biomass and underground biomass were determined[14].

3.4 Measured indicators and methods

3.4.1Plant height. The plant height was measured with a ruler, and 10 plants were determined per pot. In order to eliminate the error of the material itself, Relative plant height is used as an indicator to measure the resistance of alfalfa toF.proliferatum.

Relative plant height=Plant height of the inoculation group/Plant height of the control group ×100.

3.4.2Relative aboveground and underground biomass. The aboveground part of the alfalfa seedlings was cut off using scissors, dried in an oven at 105 ℃ for 30 min, dried at 80 ℃ to constant weight (12 h) and weighed. The total dry weight of the aboveground part of all plants in each pot was taken as the aboveground biomass. After the measurement of the aboveground biomass, the remaining roots in each pot were digged out, rinsed with running water, dried at 80 ℃ to constant weight (24 h), and weighed.

Relative aboveground biomass=Aboveground biomass of the inoculation group/Aboveground biomass of the control group ×100.

Relative underground biomass=Underground biomass of the inoculation group/Underground biomass of the control group ×100.

3.4.3Classification criteria of root disease and division criteria of response types. Referring to the classification criteria of Huang Ning[15]and Li Minquan[3], the disease in the root collar was divided into 5 levels (Table 1). Referring to the classification criteria of Pan Longqi[14]and Li Minquan[3], the response types of the root disease were defined (Table 2).

Table 1 Classification criteria for disease in root collar of alfalfa

Table 2 Classification criteria for response types of root disease

Incidence rate (%)=Number of diseased plants/Total number of plants ×100.

Disease index (%)=∑(Number of diseased plants at each level × Representative value of each level)/(Total number of plants × Representative value of the highest level)×100.

3.5 Statistical analysisExperimental data were processed using Microsoft Excel and subjected to single factor analysis of variance using SPSS 22.0 software. Multiple comparisons among the groups were performed usingLSDmethod. The significance level was set atP<0.05.

4 Results and analysis

4.1 Effect of different inoculation methods ofF.proliferatumon relative plant height of Longmu 801 and TG4Three kinds of inoculation methods were used to inoculateF.proliferatumto two varieties of alfalfa, Longmu 801 and TG4, and the relative plant height was calculated after measuring the plant height. The results are shown in Fig.1. As shown in Fig.1, there was no significant difference in the relative plant height of alfalfa among the three inoculation methods (P>0.05), and the relative plant heights of the three inoculation methods were all close to 100, indicating that different inoculation methods had no significant effect on the effect ofF.proliferatumon the plant height of Longmu 801. Under the condition of inoculation with rice grain-based inoculum at the bud stage, the relative plant height of TG4 was 51.9, significantly smaller than those under the other two inoculation methods (P<0.05). It can be seen that the effect of different inoculation methods on the relative plant height of alfalfa was related to the variety, and the effect on TG4 was greater.

4.2 Effect of different inoculation methods ofF.proliferatumon relative biomass of Longmu 801 and TG4F.proliferatumwas inoculated to Longmu 801 and TG4 with three different inoculation methods. The aboveground biomass and underground biomass were determined, and the relative aboveground biomass and relative underground biomass were calculated. The results are shown in Fig.2.

Note: Different lowercase letters on the top of the columns of the same variety indicate a significant difference at the 0.05 level.

Under the three different inoculation methods,F.proliferatumhad no significant effect on the aboveground and underground biomass of Longmu 801, and the differences among the inoculation methods were not significant (P>0.05). The relative aboveground biomass and relative underground biomass under the three inoculation methods were all close to 100, indicating that regardless of inoculation method, the biomass of Longmu 801 did not change significantly after the inoculation ofF.proliferatum. After inoculation with rice grain-based inoculum at the bud stage, the relative aboveground biomass and relative underground biomass of TG4 decreased to 36.5 and 6.1, respectively, significantly smaller than those under the other two inoculation methods (P<0.05). The differences in the relative aboveground biomass and relative underground biomass of TG4 were insignificant between the two inoculation methods of inoculation with rice grain-based inoculum at the seedling stage and inoculation by root irrigation at the seedling stage (P>0.05). It shows that the effect of different inoculation methods on the effect ofF.proliferatumon the relative biomass of alfalfa was also related to the variety, with significant effect on TG4 and insignificant effect on Longmu 801.

Fig.2 Effect of different inoculation methods on relative biomass of Longmu 801 and TG4

4.3 Pathogenicity ofF.proliferatumto Longmu 801 and TG4 under different inoculation methodsAfter inoculation withF.proliferatumat the bud stage, the incidence rates in Longmu 801 and TG4 were 73.30% and 100%, respectively, and the disease indices were both greater than 40, significantly higher than those under the other two inoculation methods. It shows that among the three inoculation methods, the effect of the inoculation with rice grain-based inoculum at the bud stage was the best. Under the same inoculation method, the incidence rate and disease index of Longmu 801 were lower than those of TG4 (Table 3). In terms of resistance level, under the three different inoculation methods, Longmu 801 was tolerant (T), immune (I) and resistant (R), higher than that of TG4 under the same inoculation method, so Longmu 801 is a disease-tolerant alfalfa variety. The incidence rate and disease index of TG4 were higher, it was susceptible to disease, its resistance level was low, and so it is a susceptible variety.

Table 3 Effect of different inoculation methods on incidence rate, disease index and resistance level of Longmu 801 and TG 4

5 Discussion

At present, there are few domestic research reports on the resistance againstFusariumspp. Ruan Liuetal.[16]isolated and identified pathogens from roots of alfalfa collected in Hebei, and a total of 71 pathogenicFusariumstrains were isolated. Among them,F.proliferatumstrain S45,F.oxysporumstrain D19-2 andFusariumsolanistrain Q1 are the most pathogenic. Li Guoliangetal.[8]inoculatedF.proliferatumto 16 alfalfa varieties at the bud stage and comprehensively evaluated their disease index, relative root length and relative seedling length. The results show that the effect ofF.proliferatumon relative root length, relative seedling length and disease index of different alfalfa varieties was significantly different (P<0.05), and the comprehensive disease resistance of varieties DS310FY, Polaris, Queen, WL319HQ and Giant-CR is weaker, and that of varieties WL168HQ, TG4, Spade and Zhaodong is strong. Cong Lilietal.[1]isolated and purified the pathogens of root rot from suspected alfalfa plants planting in Langfang of Hebei, Linhe of Inner Mongolia and Yanggao County of Shanxi, and a total of 6 strains were identified. The pathogenicity of the strains obtained was determined through the back infestation test. It is found that the pathogenicity ofF.proliferatumwas stronger, with incidence rate higher than 80%, and disease severity index higher than 60%. This is the first report thatF.proliferatumis the pathogen causing root rot in China. The results of this experiment also show thatF.proliferatumcould cause lesions in the roots of the tested alfalfa varieties, but it showed no significant effect on the relative plant height, relative aboveground biomass or relative underground biomass of Longmu 801. The reason may be related to the higher resistance of the variety. The alfalfa variety TG4 showed higher susceptibility. It shows that there is difference in the resistance of the alfalfa varieties toF.proliferatum, consistent with previous research results. Currently, the methods for evaluating the pathogenicity ofFusariumspp. to alfalfa mainly include seed inoculation method, tube seedling inoculation method, soil inoculation method, root irrigation method and root soaking method[6,17]. In this experiment, three different inoculation methods, namely inoculation with rice grain-based inoculum at the bud stage, inoculation with rice grain-based inoculum at the seedling stage, and inoculation by root irrigation a the seedling stage were used to evaluate the pathogenicity ofF.proliferatumto Longmu 801 and TG4. Under different inoculation methods, the pathogenicity ofF.proliferatumon Longmu 801 and TG4 was significantly different. The resistance of different alfalfa varieties toF.proliferatumis affected by inoculation method, inoculation and disease evaluation stage and experimental conditions, in addition to the type of strain. All these make it difficult to compare the results of pathogenic studies reported by different scholars[6,13]. As other crops, different traits of alfalfa may show different degrees of resistance to the same disease. The disease resistance coefficients of different indices of the same material are not completely consistent, and there is even a large gap. Therefore, using any single index to evaluate the resistance of alfalfa toFusariumspp. is one-sided and unreliable, and multiple indices should be used for comprehensive evaluation to be more reliable[18-19]. In this study, the indices closely related to disease resistance, namely, incidence rate, disease index, plant height, aboveground and underground biomass were used to evaluate the resistance againstF.proliferatumcomprehensively. At present, the determination of the pathogenicity ofFusariumspp. on alfalfa is mainly focused on the pathogenicity of single strains in indoor pot cultivation, while there are few reports on the pathogenicity of compound infection of multipleFusariumstrains, and further research is needed in the future[13].

6 Conclusions

Among the three inoculation methods, the pathogenicity ofF.proliferatuminoculated with rice grain-based inoculum at the bud stage was the most pathogenic to the alfalfa varieties Longmu 801 and TG4, significantly reducing the relative plant height, relative aboveground biomass and relative underground biomass of TG4. Under the inoculation method, the incidence rates and disease indices of root rot in Longmu 801 and TG4 were significantly higher than those of the other two methods. Judging from the disease resistance of the two varieties, Longmu 801 is significantly better than TG4 in disease resistance.