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Heliyon
Heliyon
Heliyon
2405-8440
Elsevier

S2405-8440(24)12631-X
10.1016/j.heliyon.2024.e36600
e36600
Research Article
Seed priming with Metarhizium anisopliae (MetA1) improves physiology, growth and yield of wheat
Bhuiyan Ashkar-Ul-Alam a
Chowdhury Md Zahid Hasan a
Mim Mahjabin Ferdaous a
Siddique Shaikh Sharmin b
Haque Md Ashraful a
Rahman Md Sazzadur c
Islam Shah Mohammad Naimul naimul@bsmrau.edu.bd
a⁎
a Institute of Biotechnology and Genetic Engineering, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, 1706, Bangladesh
b Department of Plant Pathology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur, 1706, Bangladesh
c Plant Physiology Division, Bangladesh Rice Research Institute (BRRI), Gazipur, 1701, Bangladesh
⁎ Corresponding author. naimul@bsmrau.edu.bd
23 8 2024
15 9 2024
23 8 2024
10 17 e3660029 5 2024
12 8 2024
19 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Microorganisms offer a sustainable way to increase crop production and promote eco-friendly farming. The endophytic fungus Metarhizium anisopliae is known for its multiple roles in plant ecosystems, including plant protection, symbiosis, and abiotic stress mitigation. In this study, we evaluated the potential of seed priming with M. anisopliae isolate MetA1 (MA) to enhance germination, photosynthetic efficiency, growth, and yield of two wheat varieties, BARI Gom 26 (BG26) and BARI Gom 33 (BG33) under field conditions. The study demonstrated that MA seed priming significantly improved wheat germination (by 13% and 26.04%) of BG26 and BG33, respectively. Overall, photosynthetic performance, indicated by increased leaf angle, leaf thickness, relative chlorophyll content, and linear electron flow (LEF), quantum yield of Photo System II (Phi2) was increased in MA primed wheat plants, while reducing non-photochemical quenching like NPQt, PhiNO, PhiNPQ of both varieties. These enhancements were attributed to increased shoot biomass (by 215.64% for BG26 and 280.38% for BG33), root biomass (by 141.79% for BG26 and 207.4% for BG33), effective tiller percentage (by 9.17% for BG26 and 5.7% for BG33), spike length (by 25.05% for BG26 and 25.42% for BG33), grain yield parameters such as filled grain percentage (by 23.8% for BG26 and 12.5% for BG33), and grain weight per plant (by 168.62% for BG26 and 119.62% for BG33). The findings of the research demonstrated the potential of M. anisopliae for field use in an agricultural setting, providing a sustainable means of increasing food production.

Highlights

• Metarhizium anisopliae (MA) seed priming increased wheat germination in field.

• MA regulated photosynthetic state in wheat.

• MA increased growth and yield of wheat varieties in field condition.

Keywords

Germination
Growth
Photosynthesis
Metarhizium anisopliae
Wheat
Yield
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pmc1 Introduction

Addressing the challenge of meeting global food demand amidst inevitable climate change is a pressing concern for agriculture. As climate change continues to be a major issue in the twenty-first century, we must find ways to feed our growing population. With agricultural land becoming increasingly scarce, boosting crop yields to meet food demands is crucial. Traditionally, this has involved the use of more chemical fertilizers and pesticides, but this approach is not sustainable. We need to rethink how we enhance crop growth. Rather than relying solely on these conventional methods, it is essential to explore innovative strategies that can improve crop development and productivity [1,2].

Wheat is one of the most important cereal crops in the world. It has been used in the marketing years 2021–2022, utilizing approximately 787.4 million metric tons, demonstrating a steady increase in wheat consumption worldwide [3]. More than two-thirds of the wheat produced worldwide is used for human consumption, whereas the remaining fifth is used as animal feed. However, there is a wide range in the annual per capita consumption of wheat, from 170 kg in Central Asia to 27 kg in Eastern and Southern Africa [4]. In China and India, 17–18 % of the world's wheat is consumed [5]. However, the average daily food energy input from wheat per capita in these nations is approximately 500 kcal because of their wide and varied dietary choices [6]. Wheat output must be doubled to meet the growing demand brought on by the world's population growth, which is expected to reach 9 billion people by 2050 [7].

Endophytes are fungi or bacteria that inhabit plant tissues in a symbiotic manner, offering protective benefits to plants against herbivorous pests and pathogens while enhancing the growth of plants such as grasses and wheat [8,9]. In recent decades, investigations into endophytes have revealed their substantial contribution to the promotion of plants. These endophytes have been proven to boost nutrient absorption, enhance stress tolerance, and enhance disease resistance in host plants, thereby leading to elevated crop yields. Endophytic fungi, such as Trichoderma, play a role in enhancing plant photosynthesis by triggering the upregulation of genes and pigments. When plants experience physiological or environmental stress, their photosynthetic efficiency decreases because of damage to photosystems and cellular processes caused by reactive oxygen species (ROS) [10]. The presence of these fungi in plants ultimately leads to a boost in both shoot and root growth, resulting in elevated crop yield. This, in turn, has the potential to positively impact future food production [10].

Seed priming with endophytic microbes can be a sustainable and innovative agricultural practice that aims to enhance crop performance by treating seeds before planting. This technique improves seed performance, encourages uniformity and better establishment, raises yields under a variety of conditions, strengthens resistance to environmental stress, and successfully resolves dormant problems [11]. Farmers can easily implement seed priming as a simple and cost-effective method for improving plant performance in the field. According to research results, seed priming has a positive effect on a number of linked characteristics, including stress tolerance, nutrient usage, plant growth, and seed germination [11].

A specific member of the Clavicipitaceae family, Metarhizium anisopliae (Metschn) Sorokin is a naturally occurring entomopathogenic fungus that has been extensively studied for its potential application in biological control in plant protection. It acts as a natural enemy for a variety of insect pests [[12], [13], [14], [15]]. Recent research has indicated that Metarhizium spp. not only rely on insect infestation for their propagation but also exhibit the ability to survive as saprophytic and rhizosphere-competent fungi, as well as plant endophytes. These fungi have been recognized as having dual functions as both entomopathogens and promoters of plant growth. Within the Metarhizium genus, four species have been identified as consistently surviving in soils from rhizospheric and non-rhizosphere areas of a long-term experimental farm cultivating soybean, corn, and alfalfa [16]. Metarhizium offers various benefits to host plants, including resistance to salt stress [17,18], increased plant biomass and growth [18,19], stimulation of root growth [20,21], suppression of plant pathogens [22], and assimilation of insect-derived nitrogen [23,24].

The role of Metarhizium in the growth and yield of wheat, particularly its influence on physiological characteristics under field conditions, is yet to be understood. This comprehensive study is therefore aimed at understanding the potential role of Metarhizium inoculation via seed priming in increasing germination, photosynthesis, growth and yield of wheat grown under field conditions.

2 Materials and methods

2.1 Plant and fungal material

Two high yielding wheat varieties BARI Gom 26 (BG26) and BARI Gom 33 (BG33) were obtained from the Bangladesh Wheat and Maize Research Institute, Regional Station, Jamalpur, Bangladesh. A previously collected and molecularly characterized in-house strain of M. anisopliae isolate MetA1 (MA) (GenBank: OQ581920) was used as fungal material.

2.2 Fungal inoculum preparation

The conidial inoculum was prepared for experimentation using 2-week-old cultures that had developed spores on Sabouraud Dextrose Agar Yeast (SDAY) plates at 25 ± 1 °C, previously inoculated with stock cultures. After two weeks of incubation at 25 °C, sterile 0.02% (v/v) Tween 80 was added to the culture on the surface of SDAY. The spores were then collected by gently scraping the culture with a glass hockey stick. The harvested spores, suspended in sterile 0.02% (v/v) Tween 80, were thoroughly mixed and diluted to achieve a concentration of 1 × 108 conidia/ml, and subsequently stored at 4 °C.

2.3 Field experiment

2.3.1 Land preparation

The land was plowed twice using a power tiller, and each plowing was followed by laddering. To obtain a good tilth, large clods were broken down into small pieces. All weeds and stubbles were collected and removed from the fields. The land was finally prepared three days prior to planting.

2.3.2 Experiment and treatment condition

The experiment was conducted using a randomized complete block design (RCBD). The field was divided into three blocks, and the blocks were subdivided into 4 plots: two plots for non-primed BG26 (control) and primed BG26 (treatment), and other two for non-primed BG33 (control) and primed BG33 (treatment). For each plot seeds were sown with a spacing of 25 cm × 20 cm (plant to plant, and row to row, respectively).

Healthy seeds of BG26 and BG33 were surface-sterilized using a solution of 0.5% (v/v) sodium hypochlorite and 0.2% (v/v) Tween-20 for 20 min and rinsed thoroughly 3 times with sterile distilled water (dH2O). Half of the BG26 seeds were soaked in a spore suspension of M. anisopliae for 24 h. Another half of the BG26 seeds were soaked in Tween-80 for 24 h. Similarly, half of the BG33 seeds were soaked in the spore suspension of M. anisopliae for 24 h and other half of the BG33 seeds were soaked in Tween-80 for 24 h. Subsequently, they were sown in a field. Five seeds were sown in each row, and there were ten rows in each plot. Irrigation and fertilizer were applied throughout the growing period as per requirements.

2.4 Determination

2.4.1 Germination

The number of germinated seedlings was recorded 7 days after sowing (DAS) in the field. The germination percentage was then determined.

2.4.2 Spectroscopic data

Spectroscopic measurements were conducted using a MultispeQ device (version 2.0, PhotosynQ Inc., East Lansing, MI, USA), and the relative chlorophyll content was determined using an SPAD meter [25]. Measurements were conducted at three different time points: 15, 30, and 75 days after sowing (DAS), corresponding to the early stage (ES), tillering stage (TS), and heading stage (HS) respectively in standing plot. These measurements were used to determine a number of physiological characteristics such as leaf thickness, relative chlorophyll content, linear electron flow (LEF), leaf angle, quantum yield of Photo system II (Phi2), total non-photochemical quenching (NPQt), fraction of light dedicated to non-photochemical quenching (PhiNPQ), and fraction of light lost via non-regulated photosynthesis inhibitor processes (PhiNO).

2.4.3 Plant growth and yield

After full maturity, 110 DAS, plants were carefully uprooted from the field then root were carefully washed using running water and growth parameters, viz. shoot length (SL), root length (RL), shoot fresh weight (SFW), shoot dry weight (SDW), root fresh weight (RFW), root dry weight (RDW), and yield parameters, viz. tiller number., effective tiller number., effective tiller percentage (ET percent), average spike length, spikelet/plant, filled grain/plant, unfilled grain/plant, filled grain percent (FG percent)/plant, grain weight/plant were measured. ET percentage was calculated using the following formula:ET percent (%) = (effective tiller no./ tiller no.) × 100

And FG percent/plant was calculated by the following formula:

FG Percent/plant = [filled grain (per plant)/ spikelet (per plant)] × 100

2.5 MA colonization confirmation

To assess the extent of the endophytic association (MA), three root samples per plant were collected. Each sample was surface sterilized using 70% ethanol, followed by thorough rinsing in sterile distilled water three times. Subsequently, the samples were dried on sterile paper towels and dissected to remove margins potentially contaminated by disinfectants. The remaining parts were cut into nine pieces, with each piece containing roots averaging 6 mm in length. These nine segments were placed onto Petri plates containing SDA medium supplemented with 2 mg/L of penicillin, streptomycin, and tetracycline each. The plates were covered with parafilm and incubated at 25 °C in the dark. The growth of M. anisopliae in the plant parts was observed and recorded, which facilitated the identification of M. anisopliae according to its specific traits [26].

2.6 Statistical analysis

The dataset was subjected to statistical analysis employing analysis of variance (ANOVA) and least significant difference (LSD, P < 0.05) to compare values. The analyses were conducted using RStudio version 4.3.1, dated June 16, 2023.

3 Results

3.1 Effect of MA priming on germination of wheat

The germination percentage of MA primed seed was increased by 13% and 26.04% for BG 26 and BG33, respectively, compared to non-primed seed (Fig. 1).Fig. 1 Effect of seed priming with MA on germination percentage of BG26 and BG33 wheat varieties after 7 DAS. NP = non-primed with MA, P = primed with MA. Values (means ± SEs) of each treatment were attained from three biological replications (n = 3). Different small alphabetical letter on the bars shows statistically significant differences (LSD, P < 0.05) among the values (means±SEs).

Fig. 1

3.2 Effect of MA priming on plant physiology of wheat varieties

3.2.1 Leaf angle

The leaf angle was increased by 27.9, 21.5 and 42.5% in the ES, TS, and HS stages, respectively, in the MA primed plants of BG26 compared to the non-primed plants (Fig. 2a). Similarly, the MA primed plants of BG33 also provided increased leaf angle (17.2, 30.5, and 36.8% in ES, TS, and HS stages, respectively) compared to the non-primed BG33 plants (Fig. 2a).Fig. 2 Effect of MA on parameters related to photosynthesis; a) Leaf angle, b) Relative chlorophyll, c) Leaf thickness, d) LEF, e) NPQt is the non-photochemical quenching, f) Phi2, g) PhiNO, h) PhiNPQ. NP = non-primed with MA; P = primed with MA; ES = Early stage,; TS= Tillering stage and HS= Heading stage.

Fig. 2

3.2.2 Relative chlorophyll

Relative chlorophyll was increased by 103.8, 15.36, and 20.43% in the ES, TS, and HS stages respectively, in the MA primed plants of BG26 compared to the non-primed plants (Fig. 2b). Likewise, relative chlorophyll was increased by 41.09, 20.88, and 14.74% in ES, TS and HS stages of BG33 plants compared to non-primed plants (Fig. 2b).

3.2.3 Leaf thickness

During the ES stage of BG26, plants that were primed with MA exhibited a significant increase in leaf thickness (39.14%), followed by a 37.28% rise in the TS stage and a further 18.83% increase in the HS stage, as compared to plants that were non-primed (Fig. 2c). The same pattern was observed in BG33 plants primed with MA. The leaf thickness in the ES, TS, and HS stages exhibited an increase of 40.22, 33.07, and 69.63% accordingly in plants that were primed with MA, as compared to plants that were non-primed (Fig. 2c).

3.2.4 LEF

In MA primed plants of BG26, LEF was increased by 21.88% in ES, 15.71% in TS, and 6.07% in HS compared to non-primed plants. (Fig. 2d). In the BG33 of plants primed with MA, there was an observed increase in LEF during the ES stage, TS stage, and HS stage, with respective increases of 15.89, 6.36, and 6.43%, as compared to plants that were not primed (Fig. 2d).

3.2.5 NPQt

During the ES stage, BG26 plants primed with M. anisopliae exhibited a significant reduction in non-photochemical quenching (NPQt) by 65.95%. This reduction was also observed during the TS stage by 22.25% and the HS stage by 46.20%, compared to non-primed plants (Fig. 2e). A decrease in NPQt (by 76.23, 43.37, and 77.58%) was observed in ES, TS and HS stages respectively, for MA-primed BG33 plants, compared to non-primed plants (Fig. 2e).

3.2.6 Phi2

In BG26 of MA-primed plants, an increase in phi2 was observed during the ES, TS, and HS stages, with respective increases of 204.16, 125.78, and 80.39% compared to non-primed plants (Fig. 2f). Similarly, in BG33, MA-primed plants exhibited significant increase in phi2 at the ES stage (129.42%), TS stage (52.38%), and HS stage (72.02%) compared to non-primed plants (Fig. 2f).

3.2.7 PhiNO

The PhiNO in BG26 MA primed plants was reduced by 53.9% in the ES stage, 55.69% in the TS stage, and 56.87% in the HS stage, compared to plants that were non-primed (Fig. 2g). In BG33 MA primed plants, PhiNO was reduced by 48% in the ES stage, 41.63% in the TS stage, and 62.84% in the HS stage compared to non-primed plants (Fig. 2g).

3.2.8 PhiNPQ

PhiNPQ levels exhibited a reduction of 60.34% during the ES stage, 65.48% during the TS stage, and 46.87% during the HS stage in BG26 MA primed plants, as compared to plants that were non-primed (Fig. 2h). In BG33 MA primed plants, there was a significant decrease in PhiNPQ levels, with a reduction of 50.27% in the ES stage, 39.30% in the TS stage, and 63.91% in the HS stage, as compared to plants that were non-primed (Fig. 2h).

3.3 Effect of MA priming on growth performance of wheat varieties

The seed priming with MA improved the phenotypic characters of both varieties (Fig. 3a). Shoot length significantly was increased (by 11.38 and 28.7%) in MA primed plants of BG26 and BG33, respectively, compared to the non-primed plants (Fig. 3b).Fig. 3 Effects of seed priming with MA on the growth of BG26 and BG33 wheat varieties. a) visual representation of shoot, b) shoot length, c) shoot fresh weight, d) shoot dry weight. NP = not primed with MA, P = primed with MA. Values (means ± SEs) of each treatment were attained from nine biological replications (n = 9). Different small alphabetical letter on the bars shows statistically significant differences (LSD, P < 0.05) among the values (means±SEs).

Fig. 3

The SFW was increased (by 256.03 and 200%) in MA primed plants of BG26 and BG33 respectively compared to the non-primed plants (Fig. 3c). In MA primed plants of BG26 and BG33, an increase in SDW (by 215.64 and 280.38% respectively) was observed compared to non-primed plants (Fig. 3d).

The root characteristics of both varieties showed visual improvement (Fig. 4a). However, root length of MA primed plants was not significantly different compared to non-primed plants (Fig. 4b). However, RFW was increased (by 61.39 and 284.51%) in MA primed plants of BG26 and BG33, respectively, compared to non-primed plants (Fig. 4c). Similarly, RDW was increased (by 141.79 and 207.4%) in MA primed plants of BG26 and BG33, respectively, compared to non-primed plants (Fig. 4d).Fig. 4 Effects of seed priming with MA on the growth of BG26 and BG33 wheat varieties., a) visual representation of root, b) root length, c) root fresh weight, d) root dry weight. NP = non-primed with MA, P = primed with MA. Values (means ± SEs) of each treatment were attained from nine biological replications (n = 9). Different small alphabetical letter on the bars shows statistically significant differences (LSD, P < 0.05) among the values (means±SEs).

Fig. 4

3.4 Effect of MA priming on yield and yield traits of wheat varieties

The number of tillers per plant was increased by 57.35 and 29.39% in MA primed plants of BG26 and BG33, respectively, compared to non-primed seed (Fig. 5b).Fig. 5 Effect of seed priming with MA on grain yield and yield contributing traits of wheat BG26 and BG33 wheat varieties. a) visual representation of spike, b) Tiller no./plant, c) Effective tiller no./plant, d) Effective tiller percent [ET percent (%)], e) Spike length, f) Spikelet/plant. NP = non-primed with MA, P = primed with MA. Values (means ± SEs) of each treatment were attained from nine biological replications (n = 9). Different small alphabetical letter on the bars shows statistically significant differences (LSD, P < 0.05) among the values (means±SEs).

Fig. 5

Similarly, the number of effective tillers per plant was increased by 71.5 and 36.83% in MA primed plants of BG26 and BG33, respectively, compared to non-primed plants (Fig. 5c). In MA primed plants of BG26 and BG33, effective tiller percentage was increased by 9.17 and 5.7%, respectively, compared to non-primed plants (Fig. 5d).

The improvement of spike character was observed in both varieties that were primed with MA (Fig. 5a). Spike length of MA primed plants of BG26 and BG33 was increased by 25.05 and 25.42%, respectively, compared to non-primed plants (Fig. 5e). Number of spikelets per plant was also increased when primed with MA in BG26 and BG33 by 48.21 and 57.53%, respectively, compared to non-primed plants (Fig. 5f).

3.5 Effect of M. anisopliae colonization on grain yield of wheat varieties

The grain yield showed significant improvement in MA primed varieties (Fig. 6a). In BG26 and BG33, the application of MA to plants results in a significant increase in the number of filled grains per plant (by 138.45 and 76.9%, respectively) (Fig. 6b), the percentage of filled grains (by 23.8 and 12.5%, respectively) (Fig. 6c), the weight of grains per plant (by 168.62 and 119.62%, respectively) (Fig. 6d), and a decrease in the number of unfilled grains per plant (by18.5 and 21.7%, respectively) (Fig. 6e) compared to non-primed plants.Fig. 6 Effect of MA on grain parameter of two wheat varieties. a) Visual representation of grain, b) Filled grain/plant, c) Unfilled grain/plant, d) Filled grain percent (%), e) Grain weight/plant. NP = non-primed with MA, P = primed with MA. Values (means ± SEs) of each treatment were attained from nine biological replications (n = 9). Different small alphabetical letter on the bars shows statistically significant differences (LSD, P < 0.05) among the values (means±SEs).

Fig. 6

3.6 Evaluation of root colonization of MA in wheat varieties

During early growth stages, white fungal colonies were visually observed on root sections. In later stages, olive-green colonies with characteristic green spores appeared on Sabouraud dextrose agar (SDA), confirming MA colonization.

Conversely, plants that were not primed with MA did not exhibit any fungal colonization (Supplementary data, Figs. S7a and b). Colonization rates were slightly higher for BG33 varieties compared to BG26, ranging from 33.33 to 44.44% for BG33 and 22.22 to 33.33% for BG26, as shown in Table S1 (Supplementary data).

4 Discussion

The entomopathogenic fungus M. anisopliae has been caught attention due its multifaceted functions which include both plant protection and growth promotion. In this study, we demonstrated the role of M. anisopliae isolate MetA1 (MA) seed priming on the physiology, growth and yield of wheat under field conditions. The findings are discussed below.

4.1 MA priming improved the germination of wheat

This study demonstrated that the germination rates of MA-primed (treatment) seeds were significantly higher for both wheat varieties compared to the non-primed (control) seeds. Similarly, Lastochkina et al. [27] observed an increase in germination percentages when seeds were treated with endophytes. Seed priming with endophytes can positively influence germination rates. Endophytes enhance the seed environment by improving nitrogen fixation, mineral solubilization, and producing plant hormones such as indole-3-acetic acid (IAA) and gibberellins (GA) [28]. These factors are crucial for stimulating seed germination and supporting healthy seedling growth.

4.2 MA priming improved the physiology of wheat varieties

The analysis of variance revealed that seed priming with MA (Treatment), variety (BG26 and BG33), time, (ES, TS, and HS) and their interaction significantly affected the traits: leaf angle, relative chlorophyll, leaf thickness, LEF (linear electron flow), NPQt (non-photochemical quenching), Phi2 (quantum yield of photosystem II), PhiNO, PhiNPQ (Table 1). Plants primed with MA showed increased leaf angle, LEF, Phi2, relative chlorophyll, and leaf thickness, and a decrease in PhiNO, PhiNPQ, and NPQt. Higher leaf angles can significantly influence photosynthesis in several ways. Leaves with steeper angles have a greater capacity to capture light energy and maximize photosynthesis [29].Table 1 Three-factor ANOVA for traits measured with MultispeQ in wheat plants.

Table 1Responses	Source of Variation	
Time (T)	Variety (V)	Treatment (Tr)	TxV	TxTr	VxTr	TxVxTr	
Leaf angle	***	.	***	**	***	NS	NS	
Relative chlorophyll	***	***	***	***	***	NS	NS	
Leaf thickness	***	***	***	***	***	***	***	
LEF	***	***	***	***	**	*	**	
NPQt	NS	NS	***	**	***	**	NS	
Phi2	***	***	***	***	***	***	***	
PhiNO	***	***	***	***	***	***	*	
PhiNPQ	***	***	***	*	***	***	***	
Leaf angle, Relative chlorophyll, Leaf thickness, LEF (linear electron flow), Phi2 (quantum yield of PSII), NPQt (non-photochemical quenching), PhiNPQ (fraction of light dedicated to non-photochemical quenching), PhiNO (fraction of light lost via nonregulated photosynthesis inhibitor processes).

The symbols indicate the statistical significance (NS; ., p > 0.1; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Endophytes play a vital role in boosting the chlorophyll levels in plants, especially when they are exposed to salt stress conditions [17,30]. This increased chlorophyll content in treated plants has also been reported previously as an indicator of efficient photosynthesis [31].

Enhanced leaf thickness may indicate increased cell growth or division within the leaf structure. In wild tomato (Solanum pennellii) this phenomenon has been linked to the lengthening of palisade mesophyll cells, a condition often resulting from endopolyploidy [32,33]. Such elongated palisade cells are known to facilitate better absorption of carbon dioxide (CO2) [34,35], effective light distribution across mesophyll cells [36,37], and sustainable leaf water potential under conditions of scarce water availability [38]. Therefore, enhancement of leaf thickness often serves as an adaptive advantage that enhances plant resilience by improving photosynthetic and water-use efficiency. According to Poorter et al. [39], plants often face a compromise between rapid growth and maintenance of leaf thickness, especially under conditions of limited water availability.

In Linear Electron Flow (LEF), light energy splits water molecules, leading to the flow of electrons. These electrons move through a series of reactions to form NADPH from NADP+. Therefore, increased LEF suggests that more electrons are efficiently transported through the photosynthetic electron transport chain. An increase in LEF signifies an improvement in the capacity of the plant to use the reducing agents NADPH and ATP [40], which in turn reflects increased photosynthetic efficiency.

A decrease in NPQt indicates reduced levels of non-photochemical quenching due to heat dissipation in the photosystem. In other words, this suggests that less excess light energy is dissipated as heat and more is being utilized for photosynthesis. This can be advantageous for plants because it indicates a more efficient use of light energy for photosynthetic processes, potentially leading to increased growth and productivity [41,42].

The improved efficiency of photosynthesis, as indicated by the increased Phi2 (the proportion of incoming light energy that is utilized in the photochemical processes of photosynthesis) and decreased NPQt and PhiNPQ (the proportion of incoming light energy, particularly excited electrons, which are dissipated through regulated non-photochemical quenching mechanisms) resulted in a higher chlorophyll content [43]. The light energy absorbed by chlorophyll molecules can either facilitate photosynthesis (Phi2) or be dissipated from Photosystem II (PSII) through different pathways: re-emission as heat (PhiNPQ, NPQt), or unregulated non-photochemical losses (PhiNO). These processes, involving photochemistry, heat dissipation, and non-photochemical losses, compete directly with each other directly [44,45]. In our study, an increase in Phi2 (Photochemistry II) occurred, which typically indicates a more efficient utilization of light energy for photosynthesis, which is generally considered favorable for plant growth and productivity. However, both photoprotective processes (PhiNPQ) and unregulated harmful processes (PhiNO, the proportion of incoming light energy, specifically excited electrons, which are not utilized in regulated processes of photosynthesis) decreased in primed plants, indicating less energy dissipation as heat or through other non-photochemical processes, which also contributes to the overall efficiency of photosynthesis.

4.3 MA priming improved growth performance of wheat varieties

Significant changes were observed in various vegetative characteristics, such as shoot length, shoot fresh weight (SFW), shoot dry weight (SDW), root fresh weight (RFW), and root dry weight (RDW), except for root length, in MA primed plants compared to non-primed plants (Table 2).Table 2 Statistical significance of two-way ANOVA with factors wheat variety (V) and M. anisopliae treatment (T) on different plant parameters in a randomized complete block design (RCBD).

Table 2Responses	Source of Variation	
Block (B)	Variety (V)	Treatment (T)	VxT	
Shoot Length	NS	***	***	***	
Root length	NS	NS	NS	NS	
Shoot Fresh Weight	NS	***	***	***	
Shoot Dry Weight	NS	***	***	***	
Root Fresh Weight	NS	***	***	***	
Root Dry Weight	NS	***	***	NS	
Tiller No.	NS	***	***	*	
Effective Tiller No.	NS	***	***	NS	
Effective Tiller Percentage	NS	**	***	NS	
Spike Length	NS	*	***	NS	
Spikelet/plant	NS	***	***	NS	
Filled Grain/Plant	NS	***	***	NS	
Unfilled Grain/Plant	NS	***	***	.	
FG Percentage	NS	***	***	***	
Grain Weight/Plant	NS	***	***	***	
The symbols indicate the statistical significance (NS; ., p > 0.1; *, p < 0.05; **, p < 0.01; ***, p < 0.001).

Endophytes have been shown to promote plant growth by enhancing root length and root biomass under both biotic and abiotic stress conditions [17,22,46,47]. Our findings indicate that the growth performance of wheat improved with the application of endophytes, consistent with previous research on this topic [[48], [49], [50]].

Endophytes are believed to support plant growth through various mechanisms, including the secretion of growth-promoting compounds like gibberellins, auxins, and cytokinins; the production of enzymes such as 1-aminocyclopropane-1-carboxylate deaminase, urease, and catalase; and activities like phosphate solubilization, siderophore formation, mineral solubilization, and the synthesis of indole-3-acetic acid (IAA) by endophytic fungi in the rhizosphere [[51], [52], [53], [54], [55]].

4.4 MA priming improved yield and yield traits of wheat varieties

MA primed plants exhibited significant differences in tiller number, effective tiller number, effective tiller percentage (ET%), spike length, and spikelets per plant compared to non-primed plants (Table 2). Our study supports previous research, emphasizing the crucial role of endophytic fungi in enhancing wheat yield under both abiotic and biotic stress conditions. This finding is consistent with studies by Akter et al. [50], Chowdhury et al. [17], Mimma et al. [22], Hubbard et al. [56], and Colla et al. [57], which also reported improvements in yield-contributing characteristics due to endophytic fungi.

Our research extends beyond merely observing morphological enhancements. We hypothesize that the production of various secondary metabolites, as noted by Tobina et al. [58], may further contribute to the improvement of traits that enhance wheat yield. This suggests a multifaceted role of endophytic fungi in boosting wheat production, not only by influencing physical growth parameters but also through biochemical pathways that generate beneficial compounds.

4.5 MA priming improved grain yield of wheat varieties

Plants that were primed with MA showed significant differences in terms of filled grain/plant, unfilled grain/plant, filled grain percent (%), and grain weight/plant compared to non-primed plants (Table 2). Our study corroborates earlier research, highlighting the substantial role of endophytic fungi in enhancing wheat grain yield per plant. These findings align with the studies by Chowdhury et al. [17] and Hubbard et al. [56], which also reported improvements in wheat's grain yield-related traits due to the presence of endophytic fungi.

5 Conclusions

Seed priming with Metarhizium anisopliae isolate MetA1 has shown significant potential in enhancing the germination and photosynthetic efficiency of wheat under field conditions. This method leads to improved germination rates and increased photosynthetic performance, evidenced by modifications in leaf thickness, leaf angle, relative chlorophyll content, and various photosynthetic parameters such as Phi2, PhiNO, PhiNPQ, and NPQt. Additionally, wheat plants primed with MetA1 exhibit notable increases in growth and yield, indicating the positive impact of this fungal isolate on overall crop productivity. The use of M. anisopliae as a seed priming agent offers a sustainable agricultural intervention, helping plants better withstand environmental stresses and contributing to more resilient and productive crops. This approach holds promise for broader application in other crops, promoting sustainable farming practices and improved food security. However, the performance of wheat seed priming with Metarhizium anisopliae under various soil and plant stress conditions needs to be thoroughly studied. This includes evaluating its effectiveness in enhancing wheat growth and yield under abiotic stresses such as drought, salinity, and extreme temperatures, as well as biotic stresses like pest and disease pressures.

Data availability

Data will be made available on request.

CRediT authorship contribution statement

Ashkar-Ul-Alam Bhuiyan: Writing – original draft, Investigation, Formal analysis, Data curation. Md Zahid Hasan Chowdhury: Methodology, Data curation. Mahjabin Ferdaous Mim: Investigation, Data curation. Shaikh Sharmin Siddique: Writing – review & editing, Supervision. Md Ashraful Haque: Supervision. Md Sazzadur Rahman: Resources. Shah Mohammad Naimul Islam: Writing – review & editing, Supervision, Methodology, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following is the Supplementary data to this article.Multimedia component 1

Multimedia component 1

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.heliyon.2024.e36600.
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