Effects of five mycorrhizal fungi on biomass and leaf physiological activities of walnut

Arbuscular mycorrhizal fungi (AMF) can benefit many plants, but their effects on walnuts are not yet known. The present study aimed to analyze the effect of five AMF species, namely, Acaulospora scrobiculata, Diversispora spurca, Glomus etunicatum, G. mosseae and G. versiforme on biomass production, chlorophyll contents, sugar fraction contents, and mineral element contents of walnut (Juglans regia L.) seedlings. The five AMF species colonized roots of walnut, established mycorrhizas in roots and hyphae in soil, and released easily extractable glomalin-related soil protein into soil, whilst D. spurca exhibited the best effect. All the AMF inoculations, except A. scrobiculata, stimulated shoot and root biomass production. Mycorrhizal fungal inoculations collectively increased leaf chlorophyll a, chlorophyll b, and total chlorophyll a+b concentrations, and thus promoted leaf sucrose accumulation, which provides an important mycorrhiza-carbon source to roots. AMF inoculations conferred a positive effect on leaf N, P, K, Mg, Fe, B, Zn and Cu contents, while they reduced leaf Mn contents. These results concluded that AMF were beneficial to the growth and physiological activities of walnut, which gives the support for the AMF application in walnut.


Introduction
Walnut (Juglans regia L.) is an important economy forest in many countries of the world. Walnut is often grown in mountainous areas where the soil is very poor, limiting the yield and physiological activities of walnut (Pati and Mukhopadhyay, 2009;Qin et al., 2011;Wang et al., 2016;Bu et al., 2019;Zou et al., 2019;Kong et al., 2020). To adapt to the soil environment, plants coexist with soil microorganisms such as arbuscular mycorrhizal fungi (AMF) to improve nutrient acquisition (López-Ráez et al., 2010;Latef and Chaoxing, 2011;Gill et al., 2016;Wu et al., 2019bWu et al., , 2020. AMF are a kind of soil inhabiting endophytic fungi, which form arbuscular mycorrhizal symbiosis with roots of 80% of land's plants. Arbuscular mycorrhizas are able to stimulate nutrient absorption, improve plant growth, and enhance stress tolerance of host plants (Wu et al., 2013;Adolfsson et al., 2017;He et al., 2019;Moreira et al., 2019;Rosolino et al., 2019;Zhang et al., 2020;Zou et al., 2020;Yang et al., 2021). In switchgrass plants, inoculation with Rhizophagus irregularis improved K, Mg, and Na contents in shoots (Sun and Yang, 2019). Native AMF isolated from field citrus significantly accelerated P, K, Mg, and Zn contents in leaves of trifoliate orange seedlings (Wu et al., 2019). Mathur et al. (2018) reported that R. intraradices, Funneliformis mosseae, and F. geosporum promoted the synthesis of chlorophyll probably by increasing Mg uptake. However, Glomus clarum showed a negative effect on Mn contents in sour orange (Ortas et al., 2002). Arines et al. (1990) also reported that inoculating G. mosseae or G. aggregatum significantly decreased Mn contents in red clover. As a result, more attention should be paid to the effect of AMF on nutrient uptake of host plants.
Earlier studies have shown that the survival of walnut plants in nursery was significantly improved after inoculation with G. intraradices or G. mosseae (Dolcet-Sanjuan et al., 1996). G. deserticola also improved leaf area and root weight in Juglans nigra (Dixon, 1988). In addition, inoculation with Gigaspora margarita, G. deserticola, and G. etunicatum markedly stimulated N, P, and K concentrations in leaves of J. nigra (Dixon, 1988). Behrooz et al. (2019) found that inoculation with G. mosseae and G. etunicatum alleviated drought stress of walnut. These studies indicated that AMF co-exist well with walnut, to promote its growth and to enhance stress tolerance. However, the effect of AMF on walnut has been little studied and needs to be further explored.
The aims of the present study were to evaluate the effects of five AMF species from three genera on biomass production, chlorophyll concentrations, sugar contents, and mineral element contents of walnut.

Experimental design
This experiment was conducted in completely randomized design with six inoculations with Acaulospora scrobiculata, Diversispora spurca, Glomus etunicatum, G. mosseae, G. versiforme and non-AMF control. Each treatment was replicated six times, resulting in a total of 36 pots.
Plant set-up Seeds of Juglans regia L. Liaohe 1 were sterilized with 75% alcohol for 10 min, sowed in autoclaved (0.11 MPa, 121°C, 2 h) sand, and germinated in an incubator at 28 °C/20 °C (day/night temperature) and 80% relative humidity. A month later, one seedling with uniform size and two-leaf-old was transferred into a 2.1-L plastic pot that was supplied with 2.1 kg autoclaved soil and sand (3:1, v/v). When the seedlings were transplanted, mycorrhizal fungi were inoculated. Five AMF species were Acaulospora scrobiculata Trappe, Diversispora spurca (C.M. Pfeiff., C. Walker & Bloss) C. Walker & A. Schüßler, Glomus etunicatum Becker & Gerd., Glomus mosseae (Nicolson & Gerd.) Gerd. & Trappe, and Glomus versiforme (P. Karst.) S.M. Berch, which were provided by the Bank of Glomales in China (BGC, Beijing, China). These fungi were propagated with white clover as the host plant for 3 months under potted conditions. Mycorrhizal fungal inoculums consisted of spores, sporocarps, AMF-colonized root segments, soil hyphae, and the growth substrate. For AMF inoculation, 100 g of mycorrhizal inoculum was applied to the rhizosphere of potted walnut seedlings. Non-AMF treatment received 100 g sterilized mycorrhizal inoculum. The seedlings were subsequently placed in a greenhouse with 720 μmol/m 2 /s average photon flux density, 28/20 °C day/night temperature, and 67% relative humidity from March to June, 2019.

Parameter determinations
At harvest time, the growth of walnut seedlings with different treatments showed a significant difference. The walnut seedlings were divided into the shoot and the root, and their fresh weight was measured. Subsequently, a small amount of root segments with 1-cm-long were stained according to the protocol as described by Phillips and Hayman (1970). The root AMF colonization degree was estimated as the percentage of AMF-infected root lengths versus total root lengths. Soil mycorrhizal hyphal length was determined using the procedure as outlined by Bethlenfalvay and Ames (1987). Soil easily extractable glomalin-related soil protein (EE-GRSP) was assayed by .
Leaf chlorophyll a, chlorophyll b, total chlorophyll (chlorophyll a+b), and carotenoid concentrations were calculated by Arnon (1949) using the 80% acetone solution. Leaf samples were dried to a constant weight in air oven at 75 °C for 48 and ground into 0.5 mm powder, which was used for the analysis of sugars and mineral nutrients. Leaf glucose, fructose, and sucrose contents were determined by Wu et al. (2015). The sieved leaf samples were digested by H2SO4-H2O2 and subjected to chemical analysis by an Electrochemical Analyzer (Smartchem 200) for N contents and by an ICP Specmometer (IRIS Advantage) for other mineral element contents.

Statistical analysis
Data were analyzed using the one-way analysis of variance with the SAS software (SAS Institute, Inc., Cary, NC, USA). The Duncan's Multiple Range Test at the 0.05 level was utilized to compare the significant difference among six treatments.

Changes in mycorrhizal status in roots and soils
There was not any mycorrhizal colonization found in the roots of the non-AMF-treated seedlings, while the root colonization of the seedlings inoculated with A. scrobiculata, D. spurca, G. etunicatum, G. mosseae and G. versiforme ranged from 46.0% to 76.4% (Table 1). Similarly, soil mycorrhizal hyphae were not observed in the non-AMF seedlings, but in the AMF-inoculated seedlings, varied from 1.30 m/g to 1.65 m/g. Moreover, the seedlings colonized by D. spurca, G. etunicatum, G. mosseae and G. versiforme exhibited significantly higher soil EE-GRSP concentrations, whereas A. scrobiculata did not significantly alter soil EE-GRSP concentrations, compared with non-AMF controls. Among them, mycorrhizal status in soil and root was the highest under the condition of D. spurca. As proposed by Davoodian et al. (2012), D. spurca had better compatibility with walnut seedlings than others AMF species. Previous studies have demonstrated that root AMF colonization was positively correlated with improvement of plant growth and P acquisition (Treseder and Kathleen, 2013). García-González et al. (2016) recommended soil EE-GRSP concentration as one indicator of mycorrhizal status. The correlation analysis also showed that there was a significant positive correlation between root mycorrhizal colonization and soil mycorrhizal hyphal length or soil EE-GRSP concentration (Figure 1), which was consistent with previous studies conducted by Curaqueo et al. (2010) and Wu et al. (2012). Glomalin is released into the soil to increase soil EE-GRSP concentrations for contributing nutrient cycle, when the mycorrhizal hyphae and spores senescenced or died Meng et al., 2020).

Changes in biomass production
The results of this study showed that the shoot and root biomass was positively impacted by AMF colonization (Figure 2). Compared to the non-AMF treatment, shoot and root biomass was significantly respectively increased by 54.6% and 42.9% with D. spurca, by 43.1% and 14.3% with G. etunicatum, by 22.7% and 6.5% with G. mosseae, and by 26.0% and 28.6% with G. versiforme. Meanwhile, A. scrobiculata did not significantly alter shoot and root biomass, and D. spurca exhibited the best promoted effect. Earlier results by Giri et al. (2003) showed a significant increase in shoot and root biomass in Acacia auriculiformis plants inoculated with G. fasciculatum or G. macrocarpum. In tea plants, mycorrhiza-improved plant biomass depended on AMF species (G. etunicatum, D. spurca, G. versiforme, and mixed-AMF) (Shao et al., 2018). Also, G. etunicatum, G. mosseae, and a mix-AMF had notably positive effect on biomass of walnut (Behrooz et al., 2019). Combining these previous results with our study, it can be seen that mycorrhizas could stimulate plant biomass production of walnut, dependent on AMF species, whilst D. spurca had the best effect.
Changes in leaf glucose, fructose and sucrose contents As shown in Table 2, inoculation with AMF had different effects on leaf glucose, sucrose, and fructose contents of walnut. After walnut was inoculated with A. scrobiculata, D. spurca, G. etunicatum, G. mosseae, and G. versiforme, leaf glucose contents were significantly decreased by 43.9%, 14.7%, 32.2%, 31.4% and 42.7%, and leaf fructose contents were reduced by 46.7%, 30.6%, 15.0%, 53.0% and 31.7%, respectively. Inoculation of G. mosseae did not affect the glucose content in leaves, whereas inoculation with A. scrobiculata, D. spurca, G. etunicatum, and G. versiforme significantly improved leaf sucrose contents by 65.7%, 117.5%, 69.9%, and 111.4%, respectively. It is known that AMF growth relies on the photosynthate provided by the host plant (Pfeffer et al., 2010). AMF can only absorb and utilize hexoses such as glucose from the cleavage of sucrose (Wu et al., , 2017. Our result indicated that inoculation of AMF significantly increased leaf sucrose content in walnut, and then higher sucrose content in mycorrhizal plants was conducive to the downward transport through the phloem to the root system, for the growth of AMF (Sonia et al., 2010). At the same time, AMF also dramatically reduced leaf glucose and fructose contents of walnut, because the presence of root mycorrhizal carbon pool required a large amount of glucose. Data (means ± SD, n = 4) followed by different letters above the bars indicate significant differences (P < 0.05) among treatments. Data (means ± SD, n = 4) followed by different letters above the bars indicate significant differences (P < 0.05) among treatments.
Changes in leaf nutrient contents AM symbiosis plays an important role in improving nutritional acquisition of host plants (Hodge et al., 2010). Compared with non-AMF inoculation, inoculation with different AMF species notably improved leaf K and P content (Table 3), whilst the greatest effect was found in G. etunicatum for K improvement and D. spurca for P improvement among five AMF treatments. Previous studies have shown that AMF-improved K and P contents were dependent on host plant species, AMF species, and soil environment (Diop et al., 2003;Veresoglou et al., 2011;Kilpeläinen et al., 2020). The reason why AMF promote K and P absorption of host plants may be due to the fact that mycorrhizal extraradical hyphae can extend into the soil inaccessible to the root system. In addition, AMF up-regulated mycorrhiza-specific P transporter genes to promote the P absorption by cells (Amijee et al., 1989). The mycorrhizal extraradical hyphae directly secretes phosphatase and indirectly promotes the root system to secrete organic acid to hydrolyze organophosphorus to inorganic phosphorus, all of these ways promote the uptake of P by plants (Duan et al., 2015;Perumalsamy et al., 2017).
In the present study, except that the G. mosseae treatment did not affect leaf N concentrations, the other AMF inoculations notably increased leaf N concentrations, compared to non-AMF control (Table 3). Herein, D. spurca inoculation exhibited the highest effect. A similar result is also found in the maize infected with G. intraradices, Acaulospora laevis, and Gigaspora margarita (Frey and Hannes, 1993) and white clover (Xie et al., 2020). It has been suggested that in AM plants, ammonium nitrogen was mainly absorbed by extraradical hyphae and then synthesized into arginine, which was moved to the intraradical hyphae into urea and ornithine; the ornithine was catalyzed into amino acids and then proteins in root cells (Bago et al., 2001;Govindarajulu et al., 2005;Jin et al., 2005;Johansen and Olsson, 2006;Xie et al., 2020).
All the AMF treatments significantly promoted leaf Ca, Mg, B, Fe, Zn, and Cu contents, except the reduction of Ca after G. versiforme treatment. Hereinto, D. spurca had the best promotion on Ca and B content, G. etunicatum had the strongest positive effect on Mg and Cu contents, G. versiforme had the best promotion on Fe, and G. mosseae had the best acceleration on Zn content. The important role of the soil AMF mycelium in absorbing mineral elements has been well documented (Labidi et al., 2015;Weisany et al., 2016;Tran et al., 2019;Wu et al., 2019). Earlier studies also showed that AMF had negative effects on Mn content (Arines et al., 1990;Liu et al., 2000). We also found the negative effect on leaf Mn content after AMF inoculation, except A. scrobiculata. The reduction of Mn under mycorrhization is mainly because AMs reduce the amount of manganese reductants (Liu et al., 2000). Among five AMF species, D. spurca and G. etunicatum showed better promotion on Ca, Mg, B, Fe, Zn and Cu acquisition than other AMF species, which is in line with the corresponding root mycorrhizal colonization.

Conclusions
Mycorrhizal fungi could colonize roots of walnut and also positively accelerated shoot and root biomass production and improved physiological activities with regard to leaf chlorophyll production, leaf sucrose accumulation, and leaf nutrient acquisition (e.g., P, K, Mg, B, Fe, Zn, and Cu), dependent on AMF species.
Hereinto, Diversispora spurca represented the best effect. These results provide critical support for the application of AMF in walnut in the future.