Chemicals
Three antifungal compounds including dipropyl trisulfide (DPT), dimethyl trisulfide (DMT), and 2-methyl-2-pentenal (MP) were obtained from Sigma-Aldrich (St. Louis, MO, USA). All solvents used were analytical or HPLC grade.
Plant Materials
The banana plant in this study was “Williams” (AAA genome, Cavendish subgroup) banana cultivar that is highly susceptible to Fusarium oxysporum f. sp. cubense race 4 (Warman, 2018). The six Chinese chive cultivars were collected from Fujian province (China). The cultivar Duokang Fujiu 11 (DKF) shows a strong resistance to stress and a strong special spicy odor. The Fujiubao (FJB) shows the strongest spicy odor among the six varieties but lowest stress resistance. The Fujiuhuang 2 (FJH) shows a moderate spicy odor and stress resistance, its leaves are yellow (the rest were green). The Duokang Sijiqing (DKS) shows highly resistance but almost no spicy odor. The Futaijiu 1 (FTJ) shows a strong spicy odor but weak resistance and garlic sprout. The Zigenchun Zaohong (ZGC) shows a medium level of stress resistance and spicy odor but red roots (roots of the rest are white).
Foc Fungal Materials
The original fungal inocula of Fusarium oxysporum f. sp. cubense race 4 (FOC) were kindly provided by Professor Zide Jiang of College of Agriculture at South China Agricultural University in Guangzhou (Zhang et al., 2013). The strain was maintained in Potato Dextrose Agar (PDA), and the inocula were cultured in PDA at 25 °C in darkness. PDA medium was prepared and sterilized by autoclaving at 121 °C for 20 min (Dong et al., 2019).
The FOC conidial suspension was prepared by incubating FOC in PDA broth on a shaker at 200 rpm and 28 °C for 5 days, followed by filtration with two layers of gauze to remove the mycelia. The conidial concentration was adjusted to 1 × 106 conidia per milliliter (Sun, 2019).
Inhibitory Effect Of Leaf Volatiles
To compare the inhibitory effect of the volatiles from the six Chinese chive cultivars, fresh leaves were cut into 1 cm segments or ground into powder in liquid nitrogen, respectively. Then the leaves were added in a four-room petri dish (90 mm in diameter) and inoculated with two 4 mm discs of FOC inocula. The concentration of volatiles was 0.6 g FW in the petri dish. Similar petri dish without Chinese chive leaves were used as controls. Six replicates were prepared for each treatment. Each six petri dish containing the same sample were placed in the middle of a 4,000 ml glass jar (the height and the diameters of top and bottom were 12.5, 22, and 19 cm, respectively).
The experiment was conducted in a climate-controlled room at 25 °C in darkness. Colony diameters of the inoculated FOC were measured with a Vernier caliper when the colony of the control had reached the peripheral wall.
Inhibitory Effect Of Aqueous Leachates Of The Aerial Parts
To examine the inhibitory effect of aqueous leachates of the six Chinese chive cultivars on FOC colony growth, the fresh aerial parts (50 g) were also ground into powder in liquid nitrogen and soaked in 100 mL distilled water at 25 °C for 20 min for extractions by an ultrasonic agitation, respectively ( Zuo et al 2016). Then aqueous solution was centrifuged at 10000 rpm for 5 min. After centrifugation, the supernatant was filtered first through normal filter paper (0.45 µm, Xingya Purification Factory, Shanghai, China) and then through a microfilter of pore size 0.22 µm (Whatman puradiscTM25AS polyethersulfone membrane, catalog NO.6794 − 2514, England). The final filtrate was our original leachates with a concentration of 0.5 g FW·mL− 1.
The filtered leachates (5 mL) of each cultivar and fungal culture medium (5 mL) were mixed and added in petri dishes (90 mm in diameter) and inoculated with a 4 mm discs of FOC. The concentration of final culture medium was 0.25 g FW·mL− 1. Similar petri dish without Chinese chive leachate but with the same volume of distilled water were used as controls. Six replicates were prepared for each treatment.
The experiment was conducted in a climate-controlled room at 25 °C in darkness. Colony diameters of the inoculated FOC were measured with a Vernier caliper when the colony of the control had reached the peripheral wall.
Antifungal Activities Of Dpt, Dmt And Mp
Based on our previous result, three compounds including DPT, DMT, and MP were used to test the antifungal activities against FOC colony growth. Four beakers (250 mL, 75 mm in diameter) filled with10 mL fungal culture medium of PDA were placed in a 4,000 mL glass jar (the height and the diameters of top and bottom were 12.5, 22, and 19 cm, respectively), and inoculated with 4 mm disc of FOC. A piece of degreasing cotton filled with 100 µL the antifungal compounds (DPT, DMT, and MP) was placed in the middle of the jar. The final concentration of the three compounds was 25 µL·L− 1. Similar beakers in each glass jar without antifungal compound were used as controls. The glass jars were covered carefully with two layers of cellophane to stop the volatiles from escaping the (Nandakumar et al., 2018). Five replicates were prepared for each treatment. Thus, each treatment had 20 beakers placed in 5 glass jars.
The experiment was conducted in an incubator at 25 °C. Colony diameters of the inoculated FOC were measured with a ruler from the bottom by placing the glass jar on a high platform starting from 48 h post inoculation until 5 day when the colony of the control (no antifungal compound) breakers had grown close to the peripheral wall. Quantification of DPT, DMT and MP in the volatiles or aqueous leachates
To quantify DPT, DMT and MP in the volatiles released from Chinese chive cultivars, the volatiles were trapped according to the methods described by Zhang et al (Zhang et al., 2013). The leaves of Chinese chives (150 g) were cut with 1 cm segments and added into a 500 mL flask. The volatiles were trapped through a glass tube (3 mm inner diam, 14 cm long) packed with 150 mg of Tenax TA (2.6–diphenylene oxide polymer resin) for 5 h. Trapped volatiles were eluted from each tube with 1 mL n-hexane and then were used for analysis by a gas chromatography system (GC) as described by Nandakumar, R. et al. (2018).
To quantify DPT, DMT and MP in the aqueous leachates of Chinese chive cultivars, the leaves (50 g) were ground into powder in liquid nitrogen and extracted with 100 mL distilled water at 25 °C for 20 min through an ultrasonic agitation, respectively. Then aqueous solution was centrifuged at 10000 rpm for 5 min. The supernatant (60 mL) was consecutively partitioned with dichloromethane (100 mL). The dichloromethane layer was separated with a separatory funnel, followed by evaporation under vacuum. The substance of dichloromethane phase was finally dissolved with dichloromethane to 5 mL. The final solution was filtrated by a micro filter with pore size 0.22 µm for and ready for GC analyses.
GC analyses were performed on an Agilent GC 7890B system (Agilent Technologies, Palo Alto, CA, USA) with a flame ionization detector (FID) and a capillary column Agilent 19091J-413 HP-5 (5% phenylmethyl siloxane, 30.0 m × 320 µm × 0.25 µm). The injection temperature was 150 °C, and the oven temperature was raised from 35 °C (2 min hold) to 250 °C at a rate of 10 °C/min. Two microliters of sample were injected, and nitrogen was used as the carrier gas at a flow rate of 1.0 ml/min.
Banana intercropping with three Chinese chive cultivars in the pots
Based on the results of antifungal activities and quantity of the three active compounds, three cultivars (FJH, DKS, and DKF) of Chinese chive were selected to intercrop with banana in pots (the height and the diameters were 34 and 40 cm) filled with soils from the field of monoculture banana plantation for six years in Moxi Village, Tianbao Town of Nanjing County, Fujian Province (China, 24°29’14’’ north latitude, 117°29’3’’ longitude east). Thirty days after intercropping with Chinese chive under greenhouse conditions (25 ± 4 °C; 16/8 h light/dark photoperiod), each pot was watered with 100 ml Fusarium conidial suspension (1 × 106 spores·mL− 1) prepared as described above. The monoculture banana in the same soil served as a control (CK). The treatments and controls consisted of 10 plants per replicate.
Sixty days after fungal inoculation, plant height, stem circumference, photosynthesis parameters and disease indices were recorded. Net photosynthetic rate and stomatal conductance of the bananas were measured with Li-6400 (LI-COR, Lincoln, NE, USA). At the end of the experiment, each plant was harvested to assess the disease indices (Cheng et al., 2020).
Banana intercropping with three Chinese chive cultivars in the field
Seeds of three Chinese chive cultivars (FJH, DKS, and DKF) were sown in an experimental field in Moxi Village, Tianbao Town of Nanjing County, Fujian Province (China), in March of 2016. Ten months after planting, the Chinese chive plants were then transplanted (at a spacing of 15 × 10 cm2) in a Fusarium wilt disease frequently-occurred field in the village in January of 2017. Before the chives were planted, the field was divided into 20 plots each with a size of 2.5 × 25 m2 (Yu 1999; Huang et al., 2016). Then, ten plants (at a spacing of 2.5 × 2.25 m2) of banana cultivar “Williams” were intercropped with the Chinese chive cultivars (FJH, DKS, and DKF) in the field. The field experiment design was randomized complete block design with four different treatments with 5 plots as replications. The planting site was prepared and managed using established cultivation practices, without the application of either herbicide or fungicides. Plants were fertilized twice a year by using NPK (N: P2O5: KCL = 10:3:2) with dose of 3 kg·plot − 1. The incidence of Fusarium wilt disease of banana was examined every 30 days. When the banana ripened, fruit weight of each plant was weighed.
Statistical Analysis
Antifungal activities of three antifungal compounds, inhibitory effect of volatiles or aqueous leachates of the six Chinese chive cultivars on FOC were presented as inhibition rate (%) compared to control using the following formula: inhibition rate (%) = [(colony diameters of control - colony diameters of treatment) / colony diameters of control] × 100.
All data were subjected to analysis of variance using the Statistical Analysis System Program (SPSS 9.0). Each value was expressed as the mean ± the standard error (SE). Statistical significance was analyzed using Student’s t-test and one-way analysis of variance (ANOVA). The precision of the method was confirmed by least-significant difference (LSD, %). The values were considered significant when the P value was < 0.05.