Abstract: Background and Objective: White clover generally possesses shallow root systems and is strongly dependent on mycorrhizas. This study was aimed to evaluate mycorrhizal roles in plant growth, roots and chlorophyll levels in white clover (Trifolium repens). Methodology: Three arbuscular mycorrhizal fungi (AMF) species, namely, Rhizoglomus intraradices, Diversispora versiformis and Paraglomus occultum were inoculated into potted white clover. Results: After 85 days of mycorrhizal inoculation, root mycorrhizal colonization of white clover ranged from 84.6-90.5%. Inoculation with AMF significantly increased leaf numbers, leaf area, petiole length and shoot and root biomass than non-AMF inoculation. Inoculated plants with D. versiformis and P. occultum recorded significantly higher chlorophyll a, b and a+b concentrations than non-inoculated controls. Mycorrhizal white clover plants had significantly higher nodule numbers, root total length, projected area, volume and number of 1st, 2nd and 3rd order lateral roots, as compared with non-mycorrhizal plants, regardless of AMF species used. Conclusion: It can be concluded that AMF has a positive contribution to promoting plant growth, root development and chlorophyll production in white clover. Dive versiformis represented relatively better effect than the other AM fungi in white clover.
INTRODUCTION
Arbuscular mycorrhizal fungi (AMF) are a kind of beneficial microorganisms in soils, which are in a position to establish the symbiotic relationship with roots of most plants1. The AM symbiosis shows many benefits including uptake of nutrient and water, promotion of plant growth and enhancement of stressed resistance in the host plant2,3. Although experiments in the past showed a positive association between the host plant and AM fungi, the evidence regarding AM effects on root development is scarce4.
Root system morphology refers to the spatial shape and distribution of straight roots or adventitious roots in growth substances5,6. In general, root systems have plastic and susceptible from all kinds of factors7,8, such as soil microorganisms. Studies in the past had shown the AM fungal inoculation strongly stimulates the modification of root morphology in various plants9-11. In trifoliate orange, inoculation with AMF stimulated the formation of more fine roots and less coarse roots10,11. Isobe et al.9 reported that inoculation with AMF represented a significant effect on the length and the number of tap roots and first and second-order lateral roots in kidney bean. Other studies also revealed the increase in the number of second-order roots and third-order lateral roots after inoculated with AMF12,13. These results suggested that AMF has a positive effect on root morphology.
White clover (Trifolium repens L.) can be characterized by higher productivity, faster regeneration and sprawl growth, popularly known to apply to plant greening14,15. However, white clover generally possesses shallow root systems in soils and often suffers from adverse conditions16. There are many beneficial microorganisms associated with white clover, such as AMF, etc17. The aim of the present study is to analyze the effects of three AMF species on plant biomass, chlorophyll content and root morphology of white clover plants.
MATERIALS AND METHODS
Plant culture: The seeds of white clover (Trifolium repens L.) were surface-sterilized with 70% for 10 min, rinsed three times with distilled water and sown into plastic pots (10 cm in depth, 15 cm in mouth diameter and 9 cm in inner diameter) containing 1.0 kg autoclaved (121 , 0.11 Mpa, 2 h) substance of soils and sands (2:1, v/v) on May 24, 2015. Each pot had 35 seeds of white clover, which was then thinned in 30 seedlings per pot after 5 days. After the time of sowing, 3000 spores of Rhizoglomus intraradices, Diversispora versiformis and Poroglomus occultum, provided by Bank of Glomeromycota in China (BGC) were mixed with the growth substance. Non-AM fungal treatment was supplied with the autoclaved mycorrhizal inoculum as the control.
The experiment consisted of four treatments in a randomized arrangement: R. intraradices, D. versiformis, P. occultum and non-AMF. Each treatment had three replicates, leading to a total of 12 pots.
Determinations of variables: The seedlings were harvested after 85 days of AM fungal treatments. All the plants were divided into shoots and roots, whose dry weight was determined after over-drying for 48 at 75°C. Each root system from these plants was scanned with an EPSON Flat-Scanner and analyzed with the WINRHIZO 2007d. The lateral root number in different orders was counted.
Root AM colonization was assessed by the protocol of Phillips and Hayman18 with trypan blue. Chlorophyll concentration was measured by Knudson et al.19 with the extraction of 80% acetone.
Statistical analysis: The data (Means±SD, n = 3) were statistically analyzed by one-way ANOVA in SAS (v8.1) and the Duncan’s multiple range tests were used to determine the significance between these treatments at the 0.05 level.
RESULTS AND DISCUSSION
Root AM colonization: White clover plants could be heavily colonized by the three AMF species, varied from 84.6-90.5% in AM fungal colonization and 4.2-9.7 cm–1 root in entry points (Fig. 1). Meanwhile, inoculation with D. versiformis showed a relatively greater AM development in roots than other AM fungal treatments.
Plant growth performance: The present study showed that the inoculated white clover plants with AMF represented better leaf number, leaf area, petiole length and shoot and root biomass than non-AM plants (Table 1). Hereinto, P. occultum had more definite influence on these traits than the other AMF species. The result is in agreement with the findings of Wu et al.20, who reported the positive effect of AMF on plant growth of peach (Prunus persica) plants. The improvement of plant growth in AM plants than in non-AM plants may be due to the fact that AMF is capable to help host plants to absorb more water and nutrients from the soil though developed extraradical haypae21,22.
| Fig. 1: | Root colonization and entry point of white clover (Trifolium repens L.) seedlings inoculated with three AM fungi (Rhizoglomus intraradices, Diversispora versiformis and Poroglomus occultum) |
| Data (Mean±SD, n = 3) followed by different letters among treatment indicate significant differences at 5% level | |
| Table 1: | Effect of three AM fungi (Rhizoglomus intraradices , Diversispora versiformis and Poroglomus occultum) on growth performance and biomass production of white clover |
| Data (Mean±SD, n = 3) followed by different letters among treatments indicate significant differences at 5% level | |
| Table 2: | Effects of three AM fungi (Rhizoglomus intraradices, Diversispora versiformis and Poroglomus occultum) on root morphological traits of white clover |
| Data (Mean±SD, n = 3) followed by different letters among treatments indicate significant differences at 5% level | |
Chlorophyll concentrations: The present results indicated that the treatment with D. versiformis and P. occultum but not R. intraradices significantly increased leaf chlorophyll a and chlorophyll a+b concentration in white clover seedlings, as compared with non-AMF inoculation (Fig. 2). In addition, all the AM treatments significantly increased chlorophyll b concentration, whereas did not significantly affect the level of leaf carotenoid, relative to non-AM treatment. The increase in leaf chlorophyll a, chlorophyll b and chlorophyll a+b under mycorrhization ranked as D. versiformis>P. occultum> R. intraradices in the decreasing order. This is consistent with the results in trifoliate orange by Wu et al.23 and beach plum by Zai et al.24. Greater chlorophyll levels in AM plants suggested the more production of photosynthates in the host plant, which would provide more substrates for growth and development of root systems and AM symbiosis25.
Root system morphology: In this study, inoculation with R. intraradices, D. versiformis and P. occultum dramatically increased root system morphological traits, including taproot length, total root length, surface area, volume and average diameter, relative to non-AMF control (Table 2, Fig. 3). In the three AMF species, R. intraradices and P. occultum had the superior effect on root morphology than D. versiformis (Fig. 3). It is in agreement with the results of Gutjahr et al.26 in rice plants (Oryza sativa).
| Fig. 2: | Effects of inoculation with three AM fungi (Rhizoglomus intraradices, Diversispora versiformis and Poroglomus occultum) on chlorophyll concentrations of white clover (Trifolium repens L.) seedlings |
| Data (Mean±SD, n = 3) followed by different letters among treatment indicate significant differences at 5% level | |
| Fig. 3: | Root morphology of white clover (Trifolium repens L.) seedlings inoculated with three AM fungi (Rhizoglomus intraradices, Diversispora versiformis and Poroglomus occultum) |
Greater root system architecture in AM plants will keep a good contact with soils, in favor of better uptake of water and nutrient27,28.
Number of lateral roots: The present study showed that all the AM treatments significantly increased the number of first, second and third-order lateral roots, compared with non-AM treatment (Fig. 4).
| Fig. 4: | Effects of inoculation with three AM fungi (Rhizoglomus intraradices, Diversispora versiformis and Poroglomus occultum) on the number of lateral roots in white clover (Trifolium repens L.) seedlings |
| Data (Mean±SD, n = 3) followed by different letters among treatment indicate significant differences at 5% level | |
Number of first-order lateral roots in AM plants were 18.68, 21.10 and 91.43% significantly higher under R. intraradices, D. versiformis and P. occultum conditions than under non-AMF conditions. Mycorrhizal plants with R. intraradices, D. versiformis and P. occultum represented 160, 192 and 181% significantly higher number of second-order lateral root than non-mycorrhizal plants. The number of third-order lateral root was increased by 70, 155 and 127% respectively inoculating with R. intraradices, D. versiformis and P. occultum compared with non-AM fungal plants. This is in agreement with the findings of earlier studies in trifoliate orange and kidney bean9,10,11. Greater later root numbers in AM plants mean the plants contacting more soils, which is important for better plant growth.
CONCLUSION
As a consequence, the results showed that inoculation with all three AMF species significantly increased plant growth and biomass, promoted the production of chlorophyll a, b and a+b and stimulated better root morphological and lateral root formation and thus affect the growth and development of the white clover. Meanwhile, D. versiformis represented relatively better effect than the other AMF species in white clover.
SIGNIFICANCE STATEMENTS
| • | This study tried to evaluate the effects of three AMF species on white clover |
| • | AMF inoculation significantly increased plant growth and shoot and root biomass |
| • | AM white clover plants had significantly higher nodule numbers, root morphological traits and number of lateral roots as compared with non-mycorrhizal plants |
| • | Inoculated plants with D. versiformis and P. occultum had higher chlorophyll a, b and a+b levels than non-inoculated controls |
ACKNOWLEDGMENT
This study was supported by the Plan in Scientific and Technological Innovation Team of Outstanding Young, Hubei Provincial Department of Education (T201604).