1. Introduction
Asparagus decline is a serious and increasing threat in asparagus-producing regions over the world [1] [2] [3] [4] . It is supposed to be caused by the contribution of both biotic (disease) factors [2] [3] and abiotic (allelopathy etc.) factors [5] [6] [7] . As biotic factors, the most common phenomenon is Fusarium crown and root rot caused mainly by Fusarium oxysporum f. sp. asparagi, Fusarium proliferatum and Fusarium redolence, etc. [2] [3] [4] [8] . Nahiyan et al. [9] demonstrated that Fusarium oxysporum f. sp. asparagi and Fusarium proliferatum are dominant in asparagus decline fields in Japan by PCR-SSCP (single-stranded conformational polymorphism) analysis. However, it is difficult to control these diseases successfully with cultural and chemical methods due to the perennial nature of the crop, the pathogens are soil-borne, and highly resistant cultivars have not been developed [4] [10] .
Abiotic factors are mainly related to the release of allelochemicals; nutrient imbalance, deterioration of soil physiochemical conditions, cultural factors, and excessive harvesting pressure were reported [1] [5] [6] [7] [11] [12] . As for the allelochemicals, caffeic acid, ferulic acid, quercetin, malic acid are reported as asparagus root origin [5] [6] [13] [14] . On the other hand, there have been many reports on Fusarium diseases or allelochemicals in concern with asparagus decline. However, it has been still unclear the interaction between Fusarium diseases and allelochemicals. Wu et al. [15] [16] [17] reported that allelochemicals of watermelon, such as coumarin, caffeic acid, ferulic acid, salicylic acid, cinnamic acid, have inhibitory effects on growth of Fusarium oxysporum f. sp. niveum in vitro. These reports are estimated only the direct effect of allelochemicals on Fusarium growth in vitro. In addition, it is still unclear the direct effect of asparagus allelochemicals on propagation of Fusarium oxysporum f. sp. asparagi, and the interaction between disease severity and allelochemicals in asparagus plants.
In this study, the interaction between Fusarium root rot and 4 allelochemicals in asparagus seedlings was estimated in vitro to clarify the relationship between biotic and abiotic factors in asparagus decline.
2. Materials and Methods
2.1. Foa Culture with Allelochemicals In Vitro
The conidia of Fusarium oxysporum f. sp. asparagi (Foa, MAFF305556) grown on PDA media was subcultured for 2 weeks on Czapec-Dox media containing NaNO3 3 g, K2HPO4 1 g, KCl 0.5 g, MgSO4・7H2O 0.5 g, FeSO4・7H2O 0.01 g, sucrose 30 g, agar 8 g・L−1 (pH 5.8). The conidia were further subcultured (106 conidiam L−1) in liquid Czapec-Dox media with or without addition of filter-sterilized allelochemicals (caffeic acid, ferulic acid, quercetin, malic acid; 0.01%, 0.1%, w/v) at 25˚C in the dark for one week by shaking culture (100 rpm). Then, the density of conidia was investigated using hemocytometer and calculated the propagation index of allelochemical-added plots to allelochemical-non-added plots. The average was calculated from 10 replications.
2.2. Plant Culture with Foa and Allelochemicals In Vitro
Seeds of asparagus (Asparagus officinalis L., “Welcome”) were axenically sown to Knop’s medium [18] with filter-sterilized allelochemicals (caffeic acid, ferulic acid, quercetin, malic acid; 0.01%, 0.1%, w/v, using ultra pure water with few ethanol) and cultured in the growth chamber (25˚C, 16 hr daylength). Four weeks after sowing, Foa (MAFF305556)-cultured PDA cubes (5 × 5 × 5 mm) were placed on the proximal part of the roots (Figure 1). Ten plants with two replications were used. Two weeks after Foa inoculation, the symptoms were categorized into 5 degrees: percentage of root lesion length to total root length in a root system: 1, less than 20%; 2, 20% - 40%; 3, 40% - 60%; 4, 60% - 80%; 5, 80% - 100%. The index (IDL; index of diseased length to total root length) was calculated by the following formula:
2.3. Statistical Analysis
The data were compared by Tukey’s test at P < 0.05. All analyses were performed
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Figure 1. Inoculation of Foa (a) and root lesions in asparagus seedlings in vitro (b) Foa; Fusarium oxysporum f. sp. asparagi. (a) Arrow is agar cube of Foa; (b) Arrow is root lesion caused by Foa inoculation.
using XLSTAT pro statistical analysis software (Addinsoft, New York).
3. Results
In the Fusarium oxysporum f. sp. asparagi (Foa) culture with allelochemicals in vitro, Foa propagation was suppressed in all the treatments; the degree of suppression differed with the combinations between allelochemicals and concentrations (Figure 2). In this case, the suppression effect of Foa propagation in 0.1% plots became higher than 0.01% plots among all the allelochemicals.
Four weeks after sowing in vitro, dry weight of asparagus seedlings differed among the plots with the combination of allelochemicals and concentrations (Figure 3). Dry weight of both shoots and roots became lower compared to control in the plots added with 0.1% and 0.01% caffeic acid, 0.1% ferulic acid, 0.01% quercetin, only dry weight of shoots in 0.1% malic acid. In this case, suppression of shoot and root length occurred in such plots (data not shown). No growth suppression in shoots and roots occurred in 0.01% ferulic acid, 0.1% quercetin and 0.1% malic acid.
Two weeks after Foa inoculation, incidence of Fusarium root rot showed 100% in most of the plots except ferulic acid with 0.01% and 0.1%, 0.1% quercetin and 0.1% malic acid (Figure 4). However, the ratio of diseased roots increased in 0.01% and 0.1% caffeic acid, 0.1% ferulic acid, 0.1% malic acidcompared to control. Index of diseased length became higher compared to control in 0.01% and 0.1% caffeic acid, 0.1% ferulic acid, 0.01% qercetin and 0.1% malic acid (Figure 5). In this case, IDL reached more than 60 in 0.1% of caffeic acid and ferulic acid.
4. Discussion
In this study, 4 allelochemicals with different concentration levels showed
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Figure 2. Influence of allechemicals on propagation of Foa (Fusarium oxysporum f. sp. asparagi; MAFF305556). 
, 0.01%, 
, 0.1%. Bars represent standard errors (n = 10). Columns denoted by different letters indicate significant difference according to Tukey’s test (P < 0.05).
suppression effect in Foa propagation in vitro. Wu et al. [15] [16] [17] [18] reported that allelochemicals in watermelon, such as caffeic acid, ferulic acid, salicylic acid, cinnamic acid, have inhibitory effects on growth of Fusarium oxysporum f. sp. niveum in vitro. In their reports, the suppression effect increased
along with the concentration of allelochemicals added. In this experiment, same phenomena occurred in the 4 asparagus allelochemicals, so that Foa growth itself might not be directly enhanced by such 4 allelochemicals.
In the present study, some allelochemical-added plots induced growth reduction in shoots and roots before Foa inoculation, and the severity of Fusarium root rot increased in such growth-restricted plots after Foa inoculation. In this case, growth reduction and increase in disease severity greatly appeared especially in caffeic acid-added plots. Miller et al. [6] reported that some allelochemicals suppressed seed germination of asparagus, Inotani et al. [19] mentioned cinamic acid, oxalic acid and ferulic acid induced lower germination rate in lettuce etc. Chaves et al. [20] reported suppression of germination and seedling growth in Cistus ladanifer differed with the kinds of eleven allelochemicals added. Gisele et al. [21] reported root growth reduction by the addition of caffeic acid in soybean (Glycine max). From these findings, the four allelochemicals used in this study are supposed to suppress asparagus growth by some physiological obstacles under allelochemical-accumulated conditions, and such growth reduction might enhance the disease severity of Fusarium root rot as an indirect effect. In addition, such effect might differ with the allelochemicals and concentrations in asparagus. From these findings, this study firstly reported asparagus allelochemicals on propagation of Fusarium oxysporum f. sp. asparagi, and the interaction between disease severity and allelochemicals in asparagus plants. On the other hand, Wu et al. [15] [16] [17] reported that several allelochemicals of watermelon inhibited the growth of Fusarium oxysporum f. sp. niveum, however, such allelochemicals stimulated the mycotoxin (mainly fusaric acid) production of Fusarium in vitro. From these reports, it is supposed that asparagus allelochemicals might also enhance mycotoxin production of Foa, resulting in the increase of disease severity in asparagus plants. Further study would be needed on this point.
In this experiment, the tested allelochemicals have been already reported as asparagus root origin or root extract-included chemicals [6] [13] [14] [19] . In addition, the chemical concentrations in this study were almost the same levels as the reports of Wu et al. [16] [17] . However, it has been still difficult to estimate the real concentrations of such allelochemicals in the practical asparagus decline fields. On the other hand, Wu et al. [22] described that some amino acids such as alanine containing in cotton root exudates promoted the growth of Verticillium dahliae. Hao et al. [23] mentioned that alanin containing in root exudates of watermelon promoted spore germination and sporulation of Fusarium oxysporum f. sp. niveum in vitro. In this study, the author established the model method to estimate the chemical effect on Fusarium propagation and bioassay method using asparagus seedlings in vitro. Further investigation using the method established in this study would be needed to clarify the relationship between other secondary metabolites including amino acids and biotic factors on asparagus decline.
5. Conclusion
In this study, 4 allelochemicals (caffeic acid, ferulic acid, quercetin, malic acid; 0.01%, 0.1%, w/v) suppressed propagation of Fusarium oxysporum f. sp. asparagi (Foa) in vitro. As for the axenic culture of asparagus seedlings with the 4 allelochemicals, dry weight of both shoots and roots became lower compared to control, and disease severity of Fusarium root rot increased in such growth-suppressed plots. The 4 allelochemicals used in this study are supposed to suppress asparagus growth, and such growth reduction might enhance the disease severity of Fusarium root rot as an indirect effect. In addition, such effect might differ with the allelochemicals and concentrations in asparagus. This study established the model method to estimate the allelochemical effect on Fusarium propagation and bioassay method using asparagus seedlings in vitro. Further investigations using these methods would clarify the relationship between chemical and biotic factors on asparagus decline.
Acknowledgements
This study was supported by Grants-in-Aid for Scientific Research (No. 21580029), Japan Society for the Promotion of Science (JSPS).