Guava plants growth and soil microbial respiration in response to Arbuscular mycorrhizal species inoculation under alkaline soil conditions

INTRODUCTION

Guava (Psidium guajava L.)  is a main horticultural crop throughout the tropical and subtropical zone (Das et al., 2017). Guava is widespread fruit in Egypt, as it is inexpensive and contains high levels of vitamin C, pectin, vitamin A, B2, and minerals such as phosphorus (P), calcium (Ca), and iron (Fe) (Ibrahim et al., 2010). Furthermore, guavas can grow in newly reclaimed soils due to their high adaptability and thrive in these soils (Ibrahim et al., 2010).

       The most common symbiosis between plants and fungi of the phylum Glomeromycota is arbuscular mycorrhizal fungi (AMF) (Karliński, 2021). Approximately 80% of plant species have a symbiosis relationship with AMF (Harley and Harley 1987). AMF increases the plant root surface and subsequently increases the absorption of P as they stimulate the mineralization of organic P (Barea et al., 2008).

      Estrada-Luna et al., (2000) discovered that guava plants treated with a mixture of Glomus diaphanum, Glomus albidum, and Glomus claroides had greater plant growth parameters, shoot contents of P, Mg, Cu, and Mo, and an increase in gas exchange, than plants non-inoculated with AMF. Das et al., (2017) found that guava plants treated with Glomus mosseae increased guava plant yield. Similarly, Quiones-Aguilar et al., (2020) AMF studied in guava trees as growth stimulants, their results revealed a differential influence on guava plant growth when treated with native AMF, with colonization percentages of AMF exceeding 60%.

         The ability of AMF species to improve plant growth varies (Tawaraya et al., 2012). Heidari and Karami, (2014) studied the effects of two AMF species (G. mosseae, and G. etanicatum) on sunflower yield, uptake of P, and oil content, under field circumstances and found that G etanicatum had the highest impact on seeds yield, P and oil content in seeds. Similarly, the impact of three species of AMF on the growth of specific vegetable crops grown in alkaline soils was investigated by Abou El Seoud et al., (2017).They found that the AMF species, such as G. irregularis, increased the plant growth and content of P in squash and tomato, while G. macrocarpium enhanced the plant growth and content of P carrot plants under low P level.

       The respiration of microorganisms in the soil (RMS) is one of the indexes of microbial activity in the soil (Biabani and Gholizadeh, 2020). It is one of the most important soil fertility indicators (Nannipieri et al., 2017). AMF increases RMS in temperate forests and agricultural soils by affecting soil CO₂ fluxes (Tomè et al., 2016). Also, Ardestani et al., (2019) found that RMS rates increased by using AMF.

       In this context, the present work aimed to investigate the influence of AMF inoculation, under alkaline soil conditions, on the RMS through the emission of CO₂, to increase soil quality and subsequently the plant growth.

MATERIALS AND METHODS

Soil preparation

     The soil sample was collected from the topsoil (0–30 cm) of Rosetta City, Bahira, Egypt. To homogenize and remove the roots and impurities from the soil sample, it was air-dried, crushed, and sieved through a two mm sieving size (No. 10 mesh) before being blended. The soil chemical properties were analyzed according to Page et al., (1982) as following: Soil texture is clay, pH (saturated soil paste extract) 7.81, EC (1:1) 0.82 dS/m, organic matter (OM) 1.72%, CaCO₃ 1.27%, available N 91 mg/kg soil, available P (Olsen) 7.8 mg/kg soil.

Plant materials

Guava seedlings were obtained from Al-Busaili Agricultural Research Station, Ministry of Agriculture and Soil Reclamation, Egypt. The seedlings were about three months old twenty centimeters high and had one branch.

Arbuscular Mycorrhizal Fungi (AMF) inoculation

        The applied AMF species were Rhizoglomus macrocarpium (GM); Rhizoglomus irregularis (GI) and Rhizoglomus fasciculatum(GF). The first and third species were gained from the department of plant nutrition, Göttingen-University- Germany. The second species was acquired from the department of plant pathology, Hanover University - Germany. These AMF species are activated in the Soil Microbiology lab - Soil and Agriculture Chemistry Dep, Faculty of Agriculture - Saba Basha, Alexandria University, Egypt.

Pot experiment

Plastic pots (20 cm in diameter and 18 cm depth) were washed, labeled, and a filter paper was placed on the bottom of all pots to prevent soil seep, then 3 kg soil was placed in each pot leaving an upper distance of about 5 cm without soil Then compact it until reaching a bulk density of 1.42 g cm^-3. One week before planting, all pots were irrigated to the volumetric moisture content of 0.25 cm³cm^-3, which equals 70% of soil field capacity. Later, seedlings of guava were transplanted in pots on March 6, 2020. Nitrogen (N) and Potassium (K) fertilizers were applied at the rate of 30 g NH4NO₃ and 20 g K₂SO₄ per seedling. The N fertilizer was applied at four equal doses at the rate of 7.5 g/60 ml water for each pot. The K fertilizer was applied before putting the soil in the pots. After that, the phosphorus fertilizer (calcium superphosphate 15.5 % P₂O₅) were applied at three levels before filling the pots to obtain P0 (zero P fertilizer); P1 (half of the recommended P fertilizer (15 g / seedling) and P2 (all the recommended P fertilizer (30 g /seedling) (Hernandes et al., 2012). In AMF pots, one week before planting, the soil was inoculated with 30 ml of AMF inoculums (Malibari et al., 1990). Also, 20 ml of AMF inoculums were added with the seedlings of guava, (in total, the rate of 500 A-mycorrhizal spores per pot). Every day, tap water was used to irrigate all the pots to achieve 70% of field capacity.

Plants harvesting and analysis

      The guava seedlings were harvested 120 days after planting (the harvest date July 7, 2020). At the harvest time, shoots of all guava seedlings were separated from roots. The guava shoots and half roots (by weight) were washed with tap water, distilled water, air dried, and over-dried at 70°C for 48 hours (Steyn, 1959) to constant weight, and the dry weight of the guava shoots and half roots were recorded, then ground in a mill and stored in a paper package for chemical analysis. Samples of plant material (shoots and half of the roots) were wet digested with H₂SO₄–H₂O₂ (Lowther, 1980). The vanadomolybdophosphoric method was used to determine the phosphorus concentration (Jackson, 1967).

Quantifying roots length (RL)

The remaining half of the plant roots were removed from the soil using a 0.5 mm screen and a stream of tap water. The roots were covered in layers of paper towels for 2-3 minutes to soak out any remaining moisture (Schenk and Barber, 1979). Three roots’ samples weighing 0.3 g fresh weight were used to calculate the (RL) using line intersect method for each pot (Tennant, 1975).

Where RL= root length, RFW = root fresh weight (g), N =sum of horizontal and vertical crossing.

AMF extraction and determination

        The root samples of guava seedlings were divided into 1 cm segments for each pot. About 100 root segments per pot were cleaned using 10% KOH at 90 °C for 15 min and acidified in 1% HCl for 10–15 min, then stained with (0.05%) trypan blue (Gemma et al., 1989). The AMF root colonization percentage was measured under the microscope at 200X magnification (McGonigle et al., 1990). The AMF chlamydospores were extracted by using the electric wet sieving method described by Brundrett et al., (1996). 50 g of soil from each pot treated with AMF was suspended in a sufficient amount of water and stirred with rod glass for 1 min. The suspension was passing through the sieve set with sizes 500, 250, 100, 40 meshes, respectively. The AM spores in the last two sieves were transferred on the nylon filter with a 45 µm pore size. The enumeration of AM spores was done for each sample three times under a light microscope with 40X magnification. The number of AMF spores / g soil was recorded.

Respiration of microbial soil (RMS)

        At 25°C, soil samples are incubated in a closed vessel. The CO₂ produced is absorbed in sodium hydroxide and titrated to determine its concentration (Isermeyer, 1952, and modified by Jaggi, 1976). Weigh 20 g of field-moist soil into three centrifuge tubes or test tubes (samples). Pipette 20ml of sodium hydroxide solution into the laboratory bottles and insert the tubes into the bottles. Close the bottles and incubate for 24 h at 25°C. Remove the tubes and add 2ml of barium chloride solution to precipitate the absorbed CO₂ as barium carbonate. Add 3-4 drops of indicator solution and titrate the remaining sodium hydroxide with dilute HCl. To prepare controls, perform the procedure without soil.

Available Phosphorus soil

          Available P was extracted from the soil at the harvest time using the NaHCO₃ (0.5 N, pH = 8.5) method (Olsen et al., 1954) and measured by ascorbic acid molybdenum blue method at Spectrophotometer wavelength 406 nm (Murphy and Riley 1962).

Statistical methods:

       The treatments as one factor were arranged in a randomized complete block design (RCBD) with six replicates, according to Gomez and Gomez (1984). The treatments were compared using Duncan’s LSD at a 5% level of probability using the CoStat computer program (CoStat, Ver. 6.311, 2005).

RESULTS

1. Plants dry weight

         At all P levels, there was no significant difference in shoot dry weight of guava seedlings treated with AMF species (GI).

At all P levels, the dry weight of all plants treated with the three species of AMF improved significantly as compared to plants not inoculated with AMF (Fig 1). In other words, the response of the plants' dry weight without AMF inoculation to increase P levels was higher than the other plants treated with AMF species. The plants' dry weight of guava seedlings without AMF at a high P level, increased by (1.64-fold) compared to the guava seedlings at the first P level (P0). Whereas the plants dry weight of guava seedlings inoculated with species of AMF GM, GF, and GI at the third P level (P2), increased by only (1.34-fold; 1.35-fold; and 1.1-fold) compared to the guava seedlings inoculated with the same AMF species respectively at the first P level.

        The greatest plant dry weight of guava seedlings was observed with(GI) species, while inoculation with the AMF species (GM) and (GF) led to higher plant dry weight than that of the control (uninoculated plants) but significantly lower than the other guava seedlings inoculated with (GI) specie. The plants' dry weight of guava seedlings inoculated with AMF species (GI) increased by (3.18-fold; 1.76-fold; and 1.82-fold) compared to the guava seedlings without AMF inoculation, with AMF species (GM), and (GF) respectively at the first P level and increased by (2.14-fold; 1.45-fold; and 1.49-fold) compared to the guava seedlings without AMF inoculation, with AMF species (GM), and (GF) respectively at the third P level.

        At each P level, there was no significant difference in plant dry weight of guava seedlings between AMF species (GM) and (GF).

Fig. (1): Plant dry weight (g/ plant) of guava seedlings as affected by P levels and inoculated with AMF species(GM); (GF), and (GI); different letters indicate a significant difference between AMF species at all P levels, small letters between shoot dry weight and capital letters between root dry weight, P≤0,05).

Root length (RL)

     As shown in (Fig 2), at the high P level, the root length (RL) of guava seedlings with and without AMF inoculation grew significantly. The RL of guava seedlings that were not inoculated with AMF had a stronger response to increasing P levels than the other plants treated with AMF species. The RL of guava seedlings without AMF species at a high P level increased by (1.94-fold) compared to the guava seedlings without AMF at the first P level (P0), whereas the RL of guava seedlings inoculated with AMF species at the third P level, increased by (1.46-fold; 1.57-fold; and 1.51-fold) only as compared to RL of guava seedlings treated with the same AMF species respectively at a low P level.

        The guava seedlings inoculated with (GI) produced the longest RL compared to the other plants treated with (GM) and (GF) at all P levels. When its values increased by (3.4-fold; 1.75-fold; and 1.9-fold) compared to RL of guava seedlings without AMF inoculation, with AMF species (GM), and (GF) respectively at low P level and increased by (2.5- fold; 1.7-fold; and 1.76-fold) compared to RL of guava seedlings without AMF inoculation, with AMF species (GM), and (GF) respectively at the third P level.

         When comparing the RL of guava seedlings treated with AMF species (GM) to the RL of guava seedlings treated with AMF species (GM) at each P level, there was no significant difference.

Fig. (2): Root length (m/ plant) of guava seedlings as affected by P levels and inoculated with AMF species (GM); (GI), and (GF); different letters indicate significant differences between AMF species at all P levels, P≤0,05).

3. Respiration of microbial soil (RMS)

    At all P levels, RMS treated with AMF species was a highly significant increase compared to non-treated soils. RMS of plants treated with AMF species(GM); (GF), and (GI) increased by (25.8-fold; 25.4-fold; and 34.3-fold) respectively compared to non-treated soil at the first P level (Fig 3).

    There was no significant difference in RMS of AMF species between the two high P levels. 

     RMS was significantly higher in AMF species (GI) compared to the AMF species(GM)and (GF) at all P levels. RMS with AMF specie(GI) increased by (1.32-fold; and 1.35-fold) compared to (GM); (GF) respectively at low P level and it was increased by (1.33-fold; and 1.4-fold) compared to (GM); (GF) respectively at high P level (Fig 3).

       There was no significant difference in RMS between plants treated with AMF species (GM) and other plants treated with AMF species (GF) at each P level (Fig 3).

Fig. (3): RMS (CO₂ mg. g^-1 dry soil.24h^-1) as affected by P levels and inoculated with AMF species(GM); (GI) and (GF); different letters indicate significant differences between AMF species at all P levels, P≤0,05).

4. AMF measurements

4.1.AMF root colonization % (RCP)

At the third P level, the RCP of all AMF species of guava seedlings lowered significantly (Fig 4). In other words, at low P levels, the RCP of AMF species (GI), (GM), and (GF) increased by around (1.47-fold; 1.44-fold; and 1.45-fold) respectively, compared to the same AMF species at high P levels.

The mycorrhizal root colonization % was significantly higher in AMF species(GI) compared to other AMF species(GMand GF) at all P levels. On the other hand, there was no significant difference at each P level between RCP of (GM and GF) species at all P levels.

Fig. (4): Mycorrhizal root colonization % as affected by P levels and inoculated with AMF species(GM); (GI), and (GF); different letters indicate significant differences between AMF species at all P levels, P≤0,05).

4.2. Number of AMF spores

  Significant difference was observed in the number of AMF spores as a result of increasing P levels at all AMF species (Fig 5). The number of AMF spores at GM, GF, and GI species at the lowest P level (P0) reduced by about (151%; 153%; and

143%) respectively as compared to the highest P level (P3).

At all P levels, the amount of AMF spores in guava plant roots of the AMF species (GI) was significantly higher than the other two AMF species (GM and GF). On other hand, at all P levels, there was not a significant difference in AMF spores’ number between the AMF species (GMand GF) (Fig 5).

Fig. (5): Number of AMF spores/50 g soil as affected by P levels and inoculated with AMF species(GM); (GI), and (GF); different letters indicate significant differences between AMF species at all P levels, P≤0,05).

Available phosphorus in both plant and soil

5.1. Plant phosphorus content

        The results clearly showed that the plant P concentration of guava seedlings inoculated and uninoculated with AMF improved with increasing P levels (Fig 6). Uninoculated guava seedlings had a stronger response to increasing P levels than the other plants inoculated with the other AMF species studied. The plant P concentration of guava seedlings uninoculated and inoculated with AMF species(GI); (GM) and (GF) at the third P level increased by (1.7-fold; 1.23-fold; 1.27-fold; and 1.19- fold) compared to uninoculated and inoculated with AMF species (GI); (GM) and (GF) respectively at the first P level (Fig 6). On the other hand, plant P concentrations of guava seedlings inoculated with all AMF species at the second P level compared to the third P level did not differ significantly.

       Among the AMF species studied, (GI) led to a highly significant in the plant P concentration of guava seedlings as compared to the other two AMF species (GM and GF) at all P levels (Fig 6). There was no significant difference in plant P content of guava seedlings between AMF species (GM and GF) at each P level (Fig 6).

Fig. (6): Shoot and root P concentration (mg P/ g.d.m.) of guava seedlings as affected by P levels and inoculated with AMF species(GM); (GI) and (GF); different letters indicate a significant difference between AMF species at all P levels, small letters between shoot P concentration and capital letters between root P concentration, P≤0,05).

5.2. Plant phosphorus uptake

       The plant P uptake of guava seedlings inoculated and uninoculated with AMF increased with increasing P levels (Fig 7). Also, the response of uninoculated guava seedlings to increase P levels was greater than that of the other plants treated with all studied AMF species. The plant P uptake of guava seedlings uninoculated and inoculated with all AMF species at the third P level increased by (2.65-fold; 1.34-fold; 1.72-fold; and 1.56-fold) compared with uninoculated and inoculated with AMF species(GI); (GM), and (GF) respectively at the first P level (Fig 7). On the other side, there was not a significant difference in P uptake of all AMF species treated guava seedlings at the second P level compared to the third P level.

       At all P levels, the AMF species (GI) led to a highly significant in the plant P uptake of guava seedlings as compared to the other two AMF species (GM and GF) (Fig 7). Also, there was not a significant difference in plant P uptake of guava seedlings treated with AMF species (GM) as compared to plant P uptake of guava seedlings treated with AMF species (GF) at all P levels.

Fig. (7): Shoot and root P uptake (mg P/ plant) of guava seedlings as affected by P levels and inoculated with AMF species(GM); (GI), and (GF); different letters indicate a significant difference between AMF species at all P levels, small letters between shoot P uptake and capital letters between root P uptake, P≤0,05).

5.3. Available phosphorus in soil

          With rising P levels, the available P in the soil of plants treated and untreated with AMF was significantly higher. (Fig 8). The available P in soil inoculated and uninoculated with AMF increased with increasing P levels. The soil available P of plants uninoculated and inoculated with AMF species(GI),(GM) and (GF) at the third P level increased by (2.35-fold; 1.38-fold; 1.48-fold; and 1.6-fold) as compared with uninoculated and inoculated with AMF species(GI); (GM) and (GF) respectively at the first P level (Fig 8).

      At the first P level, inoculated guava plants with different AMF species resulted in a highly significant increase in soil available phosphorus but responded differently to the different species of AMF. The available P in the soil of plants inoculated with AMF species(GI); (GM) and (GF) at the first P level increased by (1.85-fold; 1.5-fold; and 1.34-fold) respectively as compared to without AMF inoculation (control) at the first P level (P0). On the other side, at the third P level, there was no significant variation in soil available P of plants treated with AMF species as compared to plants without AMF inoculation.

       Among the AMF species studied, (GI) led to a highly significant increase in available soil phosphorus as compared to the other AMF species at all P levels (Fig 8). At all P levels, there was no significant difference was observed between AMF species (GM) and (GF) in soil available phosphorus. There was a significant high in soil available phosphorus of plants treated with AMF species (GI, GM, and GF) at the first P level as compared to the third P level.

Fig. (8): Available P in soil (mg P/ kg soil) as affected by P levels and inoculated with AMF species(GM); (GI) and (GF); different letters indicate significant differences between AMF species at all P levels, P≤0,05).