Azospirillum Brasilense And Zinc Rates Affecting Fungal Root Colonization and Yield of Cereal Crops in Succession Under Brazilian Cerrado Conditions

Soil and plant bene�cial microbes capitalize plant nutrition through successful colonization in roots rhizosphere which could sustainably increase cereal production. Zinc (Zn) is intensively reported a de�cient nutrient for maize and wheat production in Brazilian Cerrado, which could be sustainably managed by bene�cial microorganisms and their symbiotic association with other microorganisms like arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE). The objective of this study was to evaluate the effect of Azospirillum brasilense inoculation and residual Zn rates on root colonization and grain yield of maize and wheat in succession under Brazilian Cerrado conditions. These experiments were conducted in a randomized block design with four replications and arranged in a 5x2 factorial scheme. The treatments were consisted of �ve Zn rates (0, 2, 4, 6 and 8 kg ha -1 ) applied from zinc sulfate in maize and residual on wheat, and without and with seed inoculation of A. brasilense. Both crops were evaluated for root colonization of AMF and DSE, number of spores of AMF, quanti�cation of CO 2 -C and grain yield. Colonization by AMF and DSE were signi�cantly increased with interaction of Zn rates and inoculation treatments. The inoculation of A. brasilense favored root AMF and DSE colonization while increasing Zn rates by 4 kg ha -1 for maize and while 2 and 4 kg ha -1 Zn in residual for wheat reduced these colonizations. The inoculation did not in�uence spore production and CO 2 -C in both crops while maize-wheat yield were increased with Zn rates up to 4 kg ha -1 in edaphoclimatic condition of Brazilian Cerrado.


Introduction
Brazilian crop production system is dominated by soybean and maize cropping where maize (Zea mays L.) is the second large produced crop. Wheat (Triticum aestivum L.) yield is still low beyond consumption, therefore imported in a huge amount (Conab 2020). The cereal production system of Cerrado savannah is interrupted by several factors like soil-borne diseases, soil erosion and low nutrient and water use e ciency, rhizosphere microbiome and functional of soil existing microorganisms (Hat eld et al. 2017; Yang et al. 2020). However, cereal cultivation could be treated as a matter of food security due to their short life cycle. Cereals could also contribute to carbon (C) sequestration and soil organic matter which can improve nutrients pools, soil e ciency and productivity (Cherubin et al. 2018).
The intense weathering of tropical soils have devastating effects on organic matter and nutrients use e ciency especially Zn and therefore, can harm cereal productivity and soil fertility (Galindo et al. 2021).
Zinc de ciency is a worldwide recognized micronutrient de ciency that limit crop growth and productivity (Jalal et al. 2020b). The low availability of Zn has drastic effects enzymatic activities and protein synthesis. Zinc is one of the fundamental constituents in the synthesis of tryptophan, indole acetic acid The interaction of soil-plant-microorganisms is alternative strategy that could contribute to soil-plant health and productivity (Jalal et al. 2021). The PGPBs especially the genus Azospirillum has the ability to ght for colonization site in above and below soil parts of several cereal crops and synthesis of phytohormones (Fukami et al. 2018a; Galindo et al. 2021; Karimi et al. 2021). The combination of Azospirillum brasilense and tryptophan as a precursor of Zn can increase IAA production and other hormones involved in plant growth (Housh et al. 2021  Azospirillum sp. most probably number 1.65×10 4 CFU g − 1 soil n = 20, DTPA = diethylenetriaminepentaacetic acid The experimental area had been cultivated with annual leguminous and cereal crops for over 28 years. In addition, the area has been under no-tillage cultivation system for the last 13 years. The crop sequence prior to eld trial was fallow until 2013 and black oats (Avena strigosa Schreb.) in 2013. Maximum, average and minimum temperatures and rainfall observed during the eld trial are presented in Fig. 2.

Experimental design and treatments
The experimental was designed in a randomized complete block with four replicates, arranged in a 5 × 2 factorial scheme. The treatments were consisted of ve Zn rates (0, 2, 4, 6 and 8 kg Zn ha -1 ) applied from zinc sulphate (20% Zn and 10% S) and two seed inoculation with A. brasilense (without or with). The total area of each experimental plot was 13. Zinc rates (0, 2, 4, 6 and 8 kg ha − 1 ) were manually applied to soil surface at even distribution in maize crop. The calculated amount of fertilizer (zinc sulphate) per plot was applied in between rows at V 2 stage of maize (with two leaves completely unfolded). The experimental area was irrigated with central pivot irrigation system (14 mm) soon after side-dress Zn application to incorporate fertilizer in soil. The Zn fertilizer was not applied in wheat crop in order to analyse residual effect of treatments.
Field management

Maize
The area was broadcast applied with limestone (composed of 28% CaO and 20% MgO with an effective neutralizing power of 88%) at the rate of 2.5 Mg ha − 1 , 65 days before maize sowing. The amount of lime applied was based on initial soil analysis and base saturation to 70%, following Eq. 1.

Equation 1
Where LN = Limestone required in Mg ha -1 , CEC = cation exchange capacity, V2 = bases saturation to be achieved, V1 = current based saturation and ENP = effective neutralization power.

Measurements
The microbiological evaluations were performed by collecting four soil samples with maize or wheat roots at the depth of 0.00-0.10 m in each experimental plot. The collected roots were washed and stored in a 50% alcohol solution. One gram of root per plot was clari ed in KOH 10% and HCl 1% solution, stained with trypan blue 0.05% and stored in lactoglycerol to assess root AMF and DSE colonization (Phillips and Hayman 1970). Root colonization was determined by evaluating 100 segments of ne roots per plot.
The soil samples were homogenised and respiratory activity were determined by quantifying carbon released as CO 2 -C in 100 g fresh soil per plot, following the methodology of Anderson and Domsch (1993).
The remaining of collected soil was air-dried, sieved (2 mm) and stored at room temperature. The number of AMF spores were determined from 100 g dry soil sample per plot. The spores were separated from the soil according to methods of decantation and wet sieving (Gerdemann and Nicolson 1963), centrifugation and sucrose otation (Jenkins 1964). Acrylic plate with concentric rings were used to count the spores under a stereoscopic microscope (40x).
Grain yield was determined by spikes collection in useful lines of each maize and wheat plots. The grains were quanti ed after mechanical threshing and the data processed in kg ha − 1 to 13% (humidity).

Statistical analysis
All data were initially tested for normality using Shapiro and Wilk (1965) test and Levene's homoscedasticity test (p < 0.05) which showed the data to be normally distributed (W ≥ 0.90). The data was then analyzed by ANOVA (F test) in a 2-way factorial design with Zn application rates and A. brasilense inoculation, their interaction was considered xed effects in the model while block was considered a random variable. Mean separation was done for signi cant of main or interaction effects using Tukey test. Regression analysis was also performed to assess whether there is a linear or non-linear response to Zn rates using R software (R Development Core Team, 2015).

Treatments effect on maize
Maize root AMF colonization was higher in plants treated with inoculation of A. brasilense and 4 kg Zn ha − 1 as compared to non-inoculated plants (Fig. 3A). The Root AMF colonization responded non-linearly to increasing Zn rates in inoculated (up to 3.5 kg Zn ha − 1 ) and non-inoculated plants (up to 4.4 kg Zn ha − 1 ) (Fig. 3A). In contrast, root DSE colonization was higher in non-inoculated treatment with 2 and 6 kg Zn ha − 1 and in inoculated plants with 8 kg Zn ha − 1 (Fig. 3B). Root DSE colonization was linearly adjusted to increasing Zn rates in inoculated treatments and non-linearly (up to 4.3 kg Zn ha − 1 ) in non-inoculated treatments (Fig. 3B). Number of AMF spores and released CO 2 -C were not affected by Zn rates or inoculation ( Fig. 3C and F).
The maize grain yield was signi cantly in uenced by Zn rates and A. brasilense inoculation (Sup. Table   2), showing a non-linearly response (up to 3.8 kg Zn ha − 1 ) with increasing Zn rates (Fig. 3G). Also, it was observed that inoculated treatments provided greater grain yield (8286 kg ha − 1 ) compared to noninoculated treatments (7943 kg ha − 1 ). Inoculated plots were noted with an increase of 4.3% (Fig. 3H).

Treatments effect on wheat
Wheat root AMF colonization was higher in treatments with A. brasilense inoculation and absence of residual Zn application or with of 2 and 4 kg Zn ha − 1 as compared to non-inoculated treatments (Fig.  4A). Root AMF colonization was linearly decreased in inoculated treatments as Zn rates increased (Fig.  4A). While, root DSE colonization was higher in non-inoculated and without Zn residual treatments or with 2 and 6 kg Zn ha − 1 (Fig. 4B). Root DSE colonization responded linearly to increasing Zn rates regardless of inoculation (Fig. 4B). Number of AMF spores decreased linearly to increasing residual Zn rates (Fig.  4C). It was also observed that AMF sporulation increased (38 x 100 g dry soil) with A. brasilense inoculation as compared to non-inoculated treatments (34 x 100 g dry soil), an increase of 11.8% was observed (Fig. 4D). Released CO 2 -C responded non-linearly to increasing Zn residual rates (up to 3.6 kg Zn ha − 1 ) (Fig. 4E). In addition, released CO 2 -C was higher with A. brasilense inoculation (13.5 µg g − 1 soil) compared to non-inoculated treatments (12.7 µg g − 1 soil) with an increase of 6.3% (Fig. 4F). Wheat grain yield responded non-linearly to increasing residual Zn rates (up to 4.7 kg Zn ha − 1 ) (Fig. 4G), but was not affected by inoculation of A. brasilense (Fig. 4H).

Discussion
In the present study, the root AMF colonization was increased with 4 kg Zn ha −  The root architecture system of wheat has low root mycorrhization as observed in the present study (Sup. Table 2). The colonization of AM in wheat roots can be change during growth stages which may have in uence on nutritional demand (Ma et al. 2019). In addition, an increase in root colonization was observed from seedling to maturity stage with an increase uptake of 20 g kg − 1 of P and 2 mg kg − 1 of Zn in wheat plants. Such increases were not observed in the present study that probably resulting from already exist optimal levels of soil fertility.
The colonization of DSE in root system of wheat or maize and its interaction with other endophytic microorganisms is still need to be addressed. The DSE colonization occur simultaneously with AMF in plant roots (Ranelli et al. 2015) to deal with biotic and abiotic factors in optimum levels of soil fertility or even increasing doses of zinc that can affect colonization of this fungal group (Lugo et al. 2018). A previous study reported that Zn application may alter DSE (Exophiala pisciphila) colonization in maize root system (Li et al. 2011). The low concentration of Zn provided a non-mutualistic relationship between plant and DSE which may result in low biomass production regardless of inoculation. Although, higher doses of Zn fertilizer increased root colonization and biomass production which indicated that alteration in DSE behavior may be due to increasing Zn rates which is veri ed in the present study (Fig. 3A, B and Fig. 4A, B).
The bacterium A. brasilense has the ability to produce siderophores and other molecules like salicylic acid that may decrease mycelial growth (Kumar et al. 2018). The present study also showed a decrease in DSE colonization in roots of wheat ( Fig. 3B and 4B). However, there has not been reported any antagonistic effect between A. brasilense and DSE in root/ soil system (Newsham 2011). Despite this, Santos et al. (2017) reported a mutualistic association between these microorganisms in most situations but can also develop a pathogenic characteristic in others. The mycelia growth can be inhibited by bacteria action, generating colonization inhibitory effects as observed in the present study for wheat. However, the interactions are still unclear and there is need for further studies on the in uence of molecules produced by A. brasilense in root DSE colonization.
The CO 2 -C released in maize root did not show signi cant effect with inoculation or Zn fertilization ( Fig. 4E, F). While, inoculation of A. brasilense under residual Zn rates increased respiratory activity of wheat and thus increasing CO 2 -C (Fig. 4E, F) veri ed in present study ( Fig. 3F and 4F), which could lead to stabilized environment where a higher carbon as microorganism biomass is incorporated into soil and a low value of CO 2 -C is released lost to atmosphere.
The grain yield of maize was signi cantly different with Zn fertilization and inoculation and adjusted to a non-linear function with increasing Zn rates up to 3.8 kg ha − 1 . The initial soil Zn content medium (Table 1) which could meet plant needs and also explain the decreasing trend in productivity with further increase in Zn rates (Fig. 3G). In addition, inoculation with A. brasilense showed an increase in grain production (Fig. 3H). It is possible that A. brasilense favored the development of root system with higher absorption of nutrients and water that has a positive in uence on nutritional status of plant (Gómez-Godínez et al. 2019; Galindo et al. 2021).
The assimilation of water and nutrients to spike and shoot are directly related to plant nutritional status (Galindo et al. 2019) and therefore leading to higher grain productivity. Our results showed that grain yield of wheat was not statistically in uenced by inoculation of A. brasilense (Sup.

Conclusion
Root colonization by AMF and DSE was positively increased with interaction of Zn rates and A. brasilense inoculation via maize seeds. The inoculation of A. brasilense favored colonization by AMF and reduced by DSE at 4 kg Zn ha − 1 in maize, while 2 and 4 kg ha − 1 of residual Zn in wheat in succession. It was also concluded that A. brasilense inoculation increased maize grain yield and number of AMF spores and CO 2 -C released in wheat in succession.

Con icts of Interest
The authors declare no con ict of interest.

Consent to participate
Not applicable.

Consent for publication
Not applicable.

Con ict of interest
The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Code availability Not applicable.
2. Yang T, Siddique KHM, Liu K (2020) Cropping systems in agriculture and their impact on soil healtha review.  Table 2 is not available with this version.