Lamiacea family-Derived Endophytic fungi: Induced Tolerance to drought Stress in Thymus vulgaris plants

doi:10.21203/rs.3.rs-4711793/v1

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Research Article

Lamiacea family-Derived Endophytic fungi: Induced Tolerance to drought Stress in Thymus vulgaris plants

https://doi.org/10.21203/rs.3.rs-4711793/v1

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Background

Thymus vulgaris (T. vulgaris) is a valuable medicinal plant widely cultivated for its aromatic and medicinal properties. However, like many plants, T. vulgaris faces challenges such as drought stress, which significantly affects its growth, morphological, physiological, and biochemical processes. Understanding how endophytic fungi isolated from the Lamiaceae family influence T. vulgarisunder varying watering regimes can enhance its resilience against drought stress. The aim of this study is to assess the impact of individual and co-inoculation of three native endophytic species including Fusarium sp., Cladosporium puyae (C. puyae), and Curvularia australiensis (C. australiensis) on T. vulgaris growth parameters under different irrigation regimes in greenhouse conditions.

Results

It's been discovered that using fungal endophytes as a biological instrument that can benefit T. vulgaris under drought stress. Plants without endophytes exhibited significant reductions in growth, The levels of chlorophyll (Chl) and carotenoids under drought stress conditions. However, plants inoculated with fungal endophytes showed enhanced growth characteristics, elevated chlorophyll levels, and Carotenoid, indicating improved tolerance to drought stress. Additionally, fungal endophyte inoculation led to a considerable reduction in lipid peroxidation (MDA) levels and elevated activities of antioxidant enzymes including Glutathione Reductase (GR), Ascorbate Peroxidase (APX), Superoxide Dismutase (SOD), Peroxidase (POD), and Catalase (CAT) under drought stress situations.

under drought stress conditions, the activity of antioxidant enzymes such as Glutathione Reductase (GR), Ascorbate Peroxidase (APX), Superoxide Dismutase (SOD), Peroxidase (POD), and Catalase (CAT).

Conclusions

The findings suggest that the addition of fungal endophytes to the inoculum enhances drought tolerance in T. vulgaris by mitigating the harmful impact of drought stress on plant growth and Physiological functions. The higher activity of antioxidant enzymes and the improved redox state of glutathione are responsible for plants' greater resistance to drought.

APX

biofertilizer

drought tolerance

Endophyte

T. vulgaris

A major threat to ecosystem sustainability and agricultural productivity is drought stress, Farm plants are harmed by drought, an abiotic stressor that intensifies other abiotic stresses like heat, salinity, and disease attack and lowers agricultural productivity [1]. Drought reduces photosynthesis, growth, and biomass output in cultivated plants, but it also increases oxidative chloroplast damage and protein breakdown in plants [2]. Because organic farming has a higher soil organic carbon content and more plentiful plant symbionts than conventional farming, these factors may help increase soil water retention and aggregation and reduce crop yield losses caused by drought [3]. Drought causes morphological and physiological changes in plants, such as reduced transpiration and decreased photosynthesis, decreased root and shoot growth, altered osmotic conditions, increased production of reactive oxygen species (ROS), and accelerated plant senescence [4]. When plants are stressed by drought, they respond in different ways through several mechanisms, such as physiological, anatomical, biochemical, and molecular ones [5]. The world's utilization of medicinal plants is expanding, and as a result, there is currently a rising market for items made from medicinal herbs that are utilized in the food, health, and pharmaceutical industries ascend [6]. Because of its essences, the Lamiaceae family of plants is the largest in terms of therapeutic value according to healing research [7]. Thymus vulgaris, is a therapeutic perennial herb that is a member of the Lamiaceae family, which is one of the largest families of flowering plants in the world, with about 4,000 species and 220 genera [8]. Water scarcity in plant tissues generally inhibits the growth of several plant sections, including the roots and aerial tissues [9]. Drought stress alters the morphology of plants and impacts several physiological functions, including the stability and permeability of cell membranes, which in turn affects plant development [10]. Plants wilt as a result of cell membrane disruption that increases permeability and causes electrolyte leakage [11]. Increased membrane lipid peroxidation, including the generation of malondialdehyde (MDA), a measure for the extent of oxidative harm to lipids and membrane breakdown, is typically a sign of drought stress [15]. One of the most significant adverse impacts of drought stress is defined as a reduction in the amount of available and different nutrient intake for the plant [13]. Drought stress is linked to plant nutrition. Plants and endophytic fungus have developed mutualistic relationships [14]. Endophytes are organisms that live inside plants for at least a part of their life cycle without exhibiting any symptoms. They don't hurt their hosts, and sometimes they even provide a hand [15]. Fungal endophytes affect several characteristics of plants, such as their development and ability to resist biotic and abiotic stresses, intake of nutrients and minerals, as well as the generation of secondary compounds [16]. Using endophytic fungi as biofertilizers has the potential to be one of the most effective, economical, and environmentally friendly biological strategies for lessening the adverse consequences of abiotic stressors on crops (Rod) [17]. Another recent approach involves using endophytic fungi to modify how plants respond and adjust to abiotic stresses. Through a variety of ways, fungal endophytes influence the host plants' resistance to abiotic stress [18]. In environments with low water levels, plants require increased root development to better access soil moisture and nutrients; endophytic fungi are the best means of achieving this [19]. In drought stress, plants produce less chlorophyll because they limit the process of absorbing nutrients and inhibit the enzymes required for the synthesis of chlorophyll; Fungal symbiosis, under this stress, reveals the ability to increase the amount of chlorophyll in its host plants by enhancing their nutrient intake [20]. These compounds, which include phenolic acids, iso benzofurans, mannitol, and other carbohydrates, are antioxidant molecules that help plants withstand stress. these compounds can be produced by endophyte fungi, or they may motivate the plant to make them [21]. Water deficiency stress is a common event that significantly reduces T. vulgaris yield and growth on a global scale [22]. T. vulgaris yield under drought stress is unknown, hence it is critical to use mitigation and adaptation techniques. Although genetic tools and breeding techniques present a viable way to increase crop stress resistance, they are time-consuming and cause ethical questions. Therefore, utilizing microorganisms' capacity to promote plant growth may offer a long-term way to address the problems caused by climate change. It is commonly known that microorganisms that support plant growth are essential for reducing the negative impacts of environmental stress, especially drought [23]. As water deficit stress impacts various processes in plant growth [24] Stress tolerance is increased by microbe-mediated protective mechanisms, which also regulate photosynthetic capability, antioxidant enzyme activity, and the production of lateral roots [25]. Considering that a sizable portion of Iran's landmass is covered by areas that are semi-arid and dry, the undoubtedly impact of drought stress on plant growth, quality and yield particularly medicinal plants, as well as the significance of thyme as a medicinal plant, it is crucial to assess how drought stress affects the qualitative and quantitative changes in thyme medicinal plant species. As a result, the goal of the current investigation was to ascertain T. vulgaris responded to drought stress and endophytes in terms of morphological, physiological, and quantitative output.

Growth parameters

Significant (P < 0.001) interaction existed between drought level and endophyte colonization on the fresh weight (FW)/dry weight (DW) of the shoots (aerial parts), leaves, and roots. Elevated drought levels in several treatments caused a decline in the FW/DR weight of leaves, shoots, and roots. Whether under drought stress or not, Leaf, shoot, and root FW/DW weights were all noticeably greater in all endophyte-inoculated plants. than endophyte-free plants. Additionally, plants treated with a mixture of F₂ and F₃ showed significantly greater shoot dry weight (P < 0.001). The highest number of leaf, number of subdivision, trunk diameter, root width, and fresh weight of the roots and leaves were found in combination of F₁ and F₃ endophytes. Table 1 provides a description of the morphological parameters ANOVA analysis. A large majority of the examined parameters (P < 0.001) were found. The comparison of mean values for morphological parameters was given in Table 2. As demonstrated, there was a substantial interaction between the amount of drought and endophyte colonization on the dry weight of the shoots and roots (P < 0.001). The drought level reduced the dry weight of the shoots and roots (Table 2). Whether under drought stress or not, all endophyte-inoculated plants displayed noticeably greater shoot dry weights than endophyte-free plants. Furthermore, plants inoculated with a blend of two fungal endophytes [C. Puyae (F₂) and C.australiensis (F₃)] at the highest Drought level showed significantly higher (P < 0.001) shoot dry weight and shoot length (Table 2). In plants inoculated with a blend of two fungal endophytes (Fusarium sp. and C. Puyae), the largest quantity of root fresh weight was obtained in drought 50%FC, with 52% (5.2fold) variation in relation to the control (Table 2). Inoculated plants with C. Puyae fungal endophytes had significantly longer roots than other treatments under 50%FC drought (Table 2). In general, in comparison to the control, fungal can enhance morphological parameters in inoculated plants (Fig. 1). The plants associated with a combination of C. Puyae and C. australiensis (F₂F₃) fungal endophytes had significantly higher leaf number with 62% (6.2fold) compared with non-endophyte associated.

Table 1

ANOVA of drought × fungal endophyte (E) inoculation interaction on some *T. vulgaris* morphological traits
SOV	df	Mean of Squares
SOV	df	LF	LD	SF	SD	RF	RD	SL	RL	RW	NS	NL	TD
Drought	3	1.26^*	0.26^**	0.08^**	0.02^**	24.38^**	0.98^**	22.1^**	190.04^**	52.9^**	5.92^ns	205.02^*	0.02^**
Endophyte	6	40.2^**	4.14^**	5.62^**	0.03^**	304.77^**	9.87^**	105.43^**	406.71^**	64.7^**	95.82^**	8618.5^**	8.62^**
*DE**	18	4.06^**	0.09^**	0.01^**	0.009^**	9.96^**	0.33^**	7.07^**	47.1^**	27.5^**	9.78^**	1220.1^**	0.06^**
Error		1.32	0.05	0.33	0.11	1.53	0.11	8.81	15.1	8.3	4	54.58	0.01
F-value		19.15	19.13	25.45	21.11	27.73	21.56	22.43	6.56	15.43	8.15	55.4	22.8
c.v		9.23	8.73	8.71	12.3	11.19	9.61	6.82	8.82	6.09	9.52	10.42	2.4
LF, SF, SD, RF, RD, SL, RL, RW, NS, NL and TD means: leaf fresh weight, leaf dry weight, shoot fresh weight, shoot dry weight, root fresh weight, root dry weight, shoot length, root length and root width, number of sub division, number of leaf, trunk diameter
, *= significant at P ≤ 0.05 and P ≤ 0.01 respectively

Table 2

Means comparison of drought × fungal endophyte inoculation interaction on some *T. vulgaris* morphological traits
Treats		LF (gr)	LD (gr)	SF (gr)	SD (gr)	RF (gr)	RD (gr)	SL (cm)	RL (cm)	RW (cm)	NS (no)	NL (no)	TD (mm)
Drought (%FC)	Fungal Inoculation (CFUml^− 1)	LF (gr)	LD (gr)	SF (gr)	SD (gr)	RF (gr)	RD (gr)	SL (cm)	RL (cm)	RW (cm)	NS (no)	NL (no)	TD (mm)
D100 %FC	N	2.99 ^hi	0.38 ^no	2.51 ^p	1.03^fghi	3.03^klm	1.27^lm	5 ^k	19.5^hij	7^ijkl	14.64^gh	147^jk	0.9^k
	F₁	7.08^ef	1.67 ^kl	5.01^mno	1.26^fgh	6.77ⁱ	1.94^jkl	8.75^fghi	43.33^a	17.33^fgh	40 ^a	452.62 ^de	3.86^b
	F₂	5.53^fg	2.5^efghij	4.43 ^no	1.4^efg	5.43^ijk	2.09^jk	8^hij	35.93^bcd	29.16^abc	29.32^bcdef	252 ^hi	2.22^ij
	F₃	10.14^bcd	3.35^abc	7.41^ghij	2.71 ^d	13.42^cdef	3.86^abcd	12.66^bc	29.86^cdefg	26.5^bcde	34.64^abcdef	499.31^bcd	4.98 ^a
	F₁ + F₂	11.98 ^ab	3.44 ^ab	8.16^defghi	3.58^bc	16.59 ^b	4.13 ^ab	8.665^fghi	30.33^cdefg	24.5^cde	38.64^abc	448 ^de	3.38^bcde
	F₁ + F₃	7.38^ef	2.66^defgh	5.38^lmn	1.21^fgh	9.55 ^h	2.28^ijk	9.165^efgh	39.83 ^ab	33.33 ^a	24^fg	289.31^gh	2.78^fgh
	F₂ + F₃	10.21^bcd	2.05^hijk	7.2^hij	1.52^ef	15.49^bcd	3.27^defg	10.75 ^de	25.73^efgh	33 ^a	36^abcde	499.31^bcd	2.8^fgh
D75 %FC	N	2.12^hi	0.36 ^no	2.19 ^p	0.63^hij	2.26^lm	0.82^mn	4.3325 ^k	16.66^ij	3.33^jkl	10.64 ^h	91 ^k	0.9 ^k
	F₁	5.51^fg	1.1^lm	4.52^mno	1.04^fghi	6.05^ij	1.85 ^kl	7.5^ij	28.66^defg	22.66 ^def	28^cdef	359.31 ^f	2.32^hij
	F₂	5.32^fg	1.59 ^kl	6.97^hijk	2.57 ^d	10.15^gh	3.04^efgh	9.5^efgh	24.33^fgh	30^abc	41.32 ^a	499.31^bcd	3.13^cdef
	F₃	11.35^abc	3.15^abcd	8.98^cde	3.64^bc	14.52^bcd	3.32^cdef	12.8325^bc	25^efgh	22.66 ^def	36^abcde	466.62^cde	3.17^cdef
	F₁ + F₂	11.11^abc	3.19^abcd	8.3^defgh	3.53^bc	15.79^bc	3.69^abcde	12.165^bcd	23.5^ghi	22.83 ^def	25.32^ef	420 ^e	3.46^bcde
	F₁ + F₃	9.41^cde	2.41^efghij	7.52^fghij	2.12 ^de	13.96^bcde	3.03^efgh	8.165^ghij	32.66^bcde	31.33 ^ab	29.32^bcdef	525^bc	2.78^fgh
	F₂ + F₃	8.04 ^de	2.14^ghijk	8.79^cdefg	3.53^bc	12.81^cdefg	2.83^fghi	9.3325^efgh	24.66^efgh	30.73 ^ab	26.64 ^def	322^fg	3.1^cdefg
D50 %FC	N	1.69 ^hi	0.26 ^no	1.84 ^p	0.41 ^hi	3.16^lm	0.7^mn	4.8325 ^k	13.83 ^j	2.66 ^kl	12 ^h	98 ^k	0.75^kl
	F₁	3.82^gh	0.77^mn	4.32 ^no	0.74^fghi	4.41^ijkl	1.36^lm	6.8325 ^j	30.16^cdef	6.9^ijkl	37.32^abcd	200.62^ij	1.08 ^k
	F₂	11.8 ^ab	3.66 ^a	8.86^cdef	3.56^bc	13.24^cdef	3.67^abcde	10.165^ef	30.83^cdefg	25.33^bcde	38.64^abc	501.62 ^b	3.02^defg
	F₃	9.59^bcd	2.45^efghij	6.85^ijk	2.66 ^d	15^bcd	3.98^abc	10^ef	30.83^cdefg	17^fgh	40 ^ab	457.31^cde	3.08^cdefg
	F₁ + F₂	8.42 ^de	2.98^bcde	7.68^efghij	2.95 ^cd	19.77 ^a	4.31 ^a	9.915^ef	27.33^efgh	18.16^fg	29.32^bcdef	499.31^bcd	3.28^cdef
	F₁ + F₃	10.13^bcd	2.74^cdefg	5.83^klm	1.26^fgh	10.73^fgh	2.6^ghij	7.5^ij	27.66^efg	28.16^abcd	26.64^def	443.31 ^de	1.91 ^j
	F₂ + F₃	12.89 ^a	3.36^abc	9.95^bc	4.33 ^ab	15.54^bc	3.54^bcde	10^ef	31.8^cdef	21.16^ef	37.32^abcd	613.62 ^a	3.56^bcd
D25 %FC	N	1.42 ^hi	0.1^o	1.6 ^p	0.34ⁱ	0.32 ^m	0.32 ⁿ	4.665 ^k	12.23 ^j	2 ^l	14.64^gh	84 ^k	0.28^l
	F₁	3.74^gh	0.87^mn	3.94 ^o	0.59^hij	3.18^jklm	1.35^lm	9.165^efgh	27.26^efgh	12 ^hi	32^abcdef	312.62^fgh	2.18^ij
	F₂	8.18 ^de	1.97^ijk	6.59^jkl	2.29 ^d	11.4^efgh	3.04^efgh	11.665 ^cd	36.83^abc	24.16^cde	29.32^bcdef	457.31^cde	2.54^ghi
	F₃	11.03^abc	2.59^defgh	10.43 ^ab	4.79 ^l	10.04^gh	2.54^hijk	13.3325^b	24.83^efgh	8.16^gh	36^abcde	494.62^bcd	3.67^bc
	F₁ + F₂	8.44 ^de	2.18^ghij	7.24^hij	2.55 ^d	12.6^defg	3.57^bcde	10^ef	31^cdefg	8.66^ij	30.64^abcdef	438.62^de	2.97^efg
	F₁ + F₃	11.85 ^ab	2.8^cdef	9.19^bcd	2.9 ^cd	13.37^cdef	2.99^efgh	9.665^efg	26.83^efgh	27.66^abcd	38.64^abc	548.31 ^b	4.54 ^a
	F₂ + F₃	7.43^ef	1.91^jk	11.42 ^a	4.88 ^a	10.26^gh	1.87 ^kl	14.75 ^a	25.5^efgh	15^gh	32^abcdef	354.62 ^f	3.58^bcd

LF, SF, SD, RF, RD, SL, RL, RW, NS, NL and TD means: leaf fresh weight, leaf dry weight, shoot fresh weight, shoot dry weight, root fresh weight, root dry weight, shoot length, root length and root width, number of subdivision, number of leaf, trunk diameter.

Fusarium sp. (F₁), Cladosporium Puyae (F₂), Curvularia australiensis (F₃), individual inoculation and in combination with each other under different irrigation levels (100, 75, 50, and 25% FC). The control (N) was treated with distilled water. Means of columns followed by similar letters have no significant differences at 5% probability by LSD test

There are numerous and well-documented detrimental impacts of drought stress on plants in contemporary research [26]. Additionally, Researchers emphasized the disruption of plant physiological processes under drought stress, containing decreased stomatal conductance, reduced nutrient uptake, and increased production of free radicals [27]. These findings underscore the critical importance of enhancing plant resilience to drought stress and implementing optimal water management strategies to preserve plant health and agricultural productivity [28]. In the current project, drought stress resulting in a reduction in the the overall performance of morphological traits including leaf fresh weight (LF), leaf dry weight (LD), shoot fresh weight (SF), shoot dry weight (SD), Root fresh weight (RF), Root dry weight (RD), shoot length (SL), Root length (RL) and Root width (RW), number of sub division (NS), number of leaf (NL), Trunk diameter (TD) of thyme. Fungal endophytes are microorganisms that reside in plant tissues and are crucial for both normal and stressful plant growth. The assay indicated that the Fungal endophytes, especially C. australiensis (F3), enhanced morphological traits because when F3 combined with both C. Puyae (F2) and Fusarium sp (F1), increased thyme productivity. The reason for the improvement in morphological traits after inoculation with these endophytes is likely attributed to increased water and nutrient intake by the plant, better regulation of photosynthesis, and reduction in the production of stress-related molecules associated with drought [29]. Using biological agents, such as fungal endophytes, is one of numerous methods that can increase plant tolerance to drought stress. These agents are readily available and show greater promise [29]. The Lamiacea family is recognized to mitigate the negative effects of a wide range of stressors and makes up a distinct microbiome with a diversity of endophytes [30]. There has been less research done on this biome's endophytic fungus community. It has recently been shown that the fungal endophytes microbiome may have a role in fending off abiotic stressors [31]. Thus, the current investigation was carried out to examine the application of fungal endophytes obtained from Lamiacea family to improve drought stress endurance in T. vulgaris plants. Fungal endophytes significantly increase the ability of plants to withstand abiotic stressors, such as drought [32]. In this research, we discovered that although the plants are under stress from dryness, the inoculation of fungal endophytes enhanced growth, leading to better biomass in the roots and shoots, leaves, branches, and Plant survival rate, in comparison to the control. Possibly as a result of endophytes ability to stimulate plants to produce secondary metabolites like auxin and gibberellin hormones [33]. Similar fungal endophyte responses that promote plant development and mitigate stress have also been reported [34].

Chlorophyll and Carotenoid

ANOVA was used to examine the amounts of MDA, antioxidant enzymes, chlorophyll, and carotenoid. Tables 3 and 4 demonstrate that most of the measured parameters were significant (P < 0.001).

Table 3

ANOVA of drought × fungal endophyte (E) inoculation interaction on *T. vulgaris* photosynthesis pigment
SOV	df	M.S.
		Photosynthesis Pigments
		Chlorophyll a (mg. g^− 1 FW)	Chlorophyll b (mg. g^− 1 FW)	Total chlorophyll (mg. g^− 1 FW)	Carotenoids (mg. g^− 1 FW)
Drought	3	0.65^**	0.23^**	2.39^**	0.03^**
Endophyte	6	0.11^**	0.02^**	0.05^**	0.01^**
*DE**	18	0^**	0^**	0.01^**	0^**
Erorr		0	0	0	0
f- value		852.14	358.23	465.02	56.82
C.V		1.11	1.42	1.21	2.98
, *= significant at P ≤ 0.05 and P ≤ 0.01 respectively

Table 4

ANOVA of drought × fungal endophyte (E) inoculation interaction on some *T. vulgaris* antioxidant enzymes and Biochemical traits
S. O. V	df	M.S.
		Antioxidant enzymes					Biochemical
		SOD	APX	POD	GR	CAT	MDA
Drought	3	19742.25^**	446528.9^**	2356.17^**	4325.54^**	3862.15^**	89.95^**
Endophyte	6	24964.15^**	175698.3^**	1268.5^**	9548.14^**	9124.62^**	15.64^**
*DE**	18	1432^**	15364.5^**	135.47^**	157.36^**	223.47^**	29.98^**
Erorr		0.01	358.65	1.3	2.87	2.3	0.108
f- value		30215	178.56	274.32	371.35	389.54	112.07
C.V.		0.02	3.02	2.74	3.87	3.15	6.57
Superoxide dismutase (SOD), Ascorbate peroxidase (APX), Peroxidase (POD), Glutathione reductase (GR), Catalase (CAT), Malondialdehyde (MDA). , *= significant at P ≤ 0.05 and P ≤ 0.01 respectively

Higher levels of drought resulted in a decrease in the contents of carotenoid and chlorophyll (Table 5). Whether or not drought stress was applied, the endophyte-associated plants showed an increase in chlorophyll content (P < 0.001) (Table 5). Plants that were inoculated with a mix of two fungal endophytes (Fusarium sp. and C. australiensis) had more chlorophyll a content under 25% FC with 79.06% variations from the control group (Table 5). Fusarium sp. inoculation produced the highest increase in chlorophyll b, compared to the control 25% FC with 66.6%. The maximum overall chlorophyll content was found in the plants inoculated with a mixture of two fungal endophytes, Fusarium sp. and C. australiensis (F1 + F3) and C. Puyae (F1 + F2) individual.

Plants inoculated with Fusarium sp. (F1) and C. australiensis and Fusarium sp. (F1F3) fungal endophytes exhibited a high Carotenoid content in response to the maximum level of drought stress (25% FC), with differences of 63.15% and 68.42% from the control. (Table 5).

Table 5

Means comparison of drought × fungal endophyte inoculation interaction on some *T. vulgaris* photosynthesis pigment and antioxidant enzymes traits
Treatments		Chlorophyll a (mg. g^− 1 FW)	Chlorophyll b (mg. g^− 1 FW)	Total chlorophyll (mg. g^− 1 FW)	Carotenoids (mg. g^− 1 FW)	SOD (U mg^− 1 FW)	APX (U mg^− 1 FW)	POD (U mg^− 1 FW)	GR (U mg^− 1 FW)	CAT (U mg^− 1 FW)
Drought (%FC)	Fungal Inoculation (CFUml^− 1)	Chlorophyll a (mg. g^− 1 FW)	Chlorophyll b (mg. g^− 1 FW)	Total chlorophyll (mg. g^− 1 FW)	Carotenoids (mg. g^− 1 FW)	SOD (U mg^− 1 FW)	APX (U mg^− 1 FW)	POD (U mg^− 1 FW)	GR (U mg^− 1 FW)	CAT (U mg^− 1 FW)
D100% FC	N	0.88^h	0.66^gh	1.57^ef	0.29^jk	40.15^w	236.71ⁿ	2.77^r	7.422ⁿ	17.09^mn
	F₁	1.05^c	0.78^a	1.65 ^c	0.41^bc	106.38^t	402.32^l	22.26^o	15.882^kl	22.52^l
	F₂	1.12^a	0.77^ab	1.89^a	0.42 ^b	159.36ⁿ	442.32^k	29.76^jk	16.322^kl	32.61^jk
	F₃	1.03^c	0.71^d	1.6^de	0.35^fg	119.62^q	515.89^j	24.33^no	22.352^j	49.37^g
	F₁ + F₂	1.06^c	0.74^c	1.89^a	0.45 ^a	165.99^m	590.89^hi	25.16^mn	9.392ⁿ	30.32^k
	F₁ + F₃	1.11^ab	0.76^b	1.79^b	0.38 ^de	74.4^v	646.25^g	32.8^no	12.532^m	47.69^gh
	F₂ + F₃	0.91^g	0.67^fg	1.63^cd	0.36^ef	106.61^s	321.25^m	13.54^p	15.202^klm	18.52^m
D75% FC	N	0.69^l	0.62^j	1.49^h	0.25 ^l	119.62^q	405.18^l	8.14^q	13.232^lm	34.24^j
	F₁	0.98^e	0.74^c	1.62^cd	0.39 ^cd	232.2^g	538.03^j	43.09^ef	27.612ⁱ	57.99^d
	F₂	1.1^b	0.61^jk	1.55^fg	0.39 ^cd	212.34ⁱ	590.03^hi	44.54^de	17.622^k	62.94^ab
	F₃	0.88^h	0.6^k	1.52^gh	0.33^ghi	172.6^l	530.89^j	42.41^efg	40.562^fg	64.99^a
	F₁ + F₂	0.94^f	0.68^ef	1.64^c	0.41^bc	192.5^jk	662.32^fg	41.59^fg	13.372^lm	61.08^bc
	F₁ + F₃	0.99^e	0.64ⁱ	1.63^cd	0.35^fg	258.69^f	598.63^h	35.13ⁱ	28.632ⁱ	62.54^ab
	F₂ + F₃	0.87^h	0.61^jk	1.48^h	0.35^fg	139.49^o	554.46^ij	31.59^j	31.682^h	79.15^cd
D50% FC	N	0.5^o	0.41^p	1.04^l	0.21^mn	172.6^l	526.61^j	14.86^p	16.322^kl	31.44^k
	F₁	0.93^f	0.69^e	1.11^k	0.36^ef	291.81^e	593.75^hi	48.73^c	34.672^h	50.26^fg
	F₂	0.88^h	0.53^m	1.31ⁱ	0.35^fg	517.83^a	876.61^c	49.48^c	42.672^f	54.34^e
	F₃	0.79ⁱ	0.55^l	1.31ⁱ	0.31^ij	430.88^b	591.25^hi	57.83^b	52.672^e	51.37^f
	F₁ + F₂	0.76^jk	0.51^mn	1.22^j	0.38 ^de	364.65^d	697.32^ef	44.63^de	33.592^h	58.2^d
	F₁ + F₃	0.93^f	0.51^mn	1.2^j	0.34^fgh	397.77^c	943.39^b	38.53^h	34.402^h	58.19^d
	F₂ + F₃	0.76^jk	0.52^mn	1.29ⁱ	0.33^ghi	232.2^g	706.96^e	72.24^a	55.972^cd	51.69^f
D25% FC	N	0.43^p	0.39^q	0.78^o	0.19 ⁿ	106.38^t	681.6^efg	8.05^q	17.382^k	9.45^o
	F₁	0.69^l	0.65^hi	0.93^m	0.31^ij	225.58^h	682.32^efg	35.31ⁱ	57.702^c	4.96^p
	F₂	0.75^k	0.45^o	1.1^k	0.28 ^k	192.38^k	965.89^ab	36.17ⁱ	51.672^e	15.49ⁿ
	F₃	0.69^l	0.46^o	0.84ⁿ	0.22 ^m	132.87^p	855.89^c	51.99^d	75.232^a	40.09ⁱ
	F₁ + F₂	0.65^m	0.39^q	1.08^k	0.28 ^k	192.59^j	797.32^d	28.71^kl	53.622^de	24.02^l
	F₁ + F₃	0.77^j	0.35^r	1.09^k	0.32 ^hi	79.88^v	984.02^a	27.06^lm	38.502^g	14.59ⁿ
	F₂ + F₃	0.57ⁿ	0.38^q	0.91^m	0.29 ^k	113.73^r	740.89^d	40.58^gh	64.972^b	45.3^h

Fusarium sp. (F₁), Cladosporium Puyae (F₂), Curvularia australiensis (F₃), alone and combined inoculation at varying irrigation levels (100, 75, 50, and 25% FC). Distinctive water was applied to the control (N). Means of columns followed by similar letters have no significant differences at 5% probability by LSD test

The study's findings show that stress brought on by a water scarcity is linked to a drop in chlorophyll concentration. This decrease may indicate oxidative stress caused on by photooxidation of pigments and degradation of chlorophyll. Since chlorophyll is necessary for photosynthesis, any drop in chlorophyll concentration would have an impact on the capacity to photosynthesize. On the other hand, Compare to non-infected plants, endophyte-treated plants preserved more chlorophyll, indicating that endophytes can increase chlorophyll stability under a water scarcity. The chlorophyll rate was higher in sunchoke plants infected with endophytes [35]. Any decrease in the amount of chlorophyll would impact photosynthetic efficiency. Chlorophyll degradation and pigment photooxidation cause a drop in total chlorophyll, a, b, and carotenoids under situations of severe drought stress. Apart from maintaining and shielding the lipid phase of thylakoid membranes, carotenoids also serve as radical scavengers and singlet-oxygen quenchers [36]. The T. vulgaris plants inoculated with endophytes might be resistant to drought stress due to their increased Carotenoid content. A greater collecting of chlorophyll was observed in tomatoes inoculated with endophytes under duress [37]. Drought stress results in oxidative stress, leading to lipid bilayer peroxidation, which promotes development and modifies the function and role of cellular organelles [38]. The increased photosynthetic efficiency seen in this work is compatible with the increased chlorophyll content, stomatal conductance, and photosynthetic rate of T. vulgaris plants treated with fungal endophytes, particularly Fusarium sp. (F1) [39]. These results suggest that stress related to water deficit is related to a decline in chlorophyll content. However, compared to non-infected plants, endophyte-treated plants preserved more chlorophyll, indicating that endophytes can improve chlorophyll stability during a drought. This demonstrates how important endophytes are for preserving plant health and photosynthetic efficiency as well as for boosting plant tolerance to drought stress.

MDA content

The T. vulgaris plants' MDA contents significantly increased as a result of drought stress (Fig. 2). When drought stress was applied, a distinct response pattern was seen in the plants injoculated with fungal endophytes. The plants that were free of endophytes and 50% FC had the greatest MDA content (control). When comparing the MDA content under 50% FC to the control plants, inoculation with a combination of fungal endophytes (F1 + F2, F1 + F3, and F2 + F3) and endophyte application individually reduced the MDA content. When compared to the control, plants treated with Fusarium sp. (48.18%) showed the lowest level of lipid peroxidation (P ≤ 0.001).

Although the MDA concentration may be assessed to determine stress harm, antioxidant activity can help by scavenging reactive oxygen species (ROS) and promoting membrane integrity prior to damage developing [40]. In this study, When T. vulgaris plants were cultivated in a drought, there was an increase in the generation of MDA (lipid peroxidation). Elevated ROS levels during extreme stress auto-catalyze lipid membrane peroxidation and alter the membrane's semi-permeability [41]. Lipid peroxidation was inhibited by fungal endophyte colonization, which was minimal in contrast to the plants in control. Applications on an individual basis of Fusarium sp. and C. australiensis reduced the inoculated plants' lipid peroxidation levels in comparison to the control, presumably due to endophytes promote antioxidant responses and prevent membrane damage. This keeps oxidative damage from harming the plants. It has been shown that fungal endophytes reduced stress by lowering the degree of lipid oxidation, which is consistent with the results of the current investigation [42]. By preserving the amount of proline in the host tissue and reducing MDA, endophytes increase plant resistance to water deficiency stress [43]. Therefore, our research indicated that plants inoculated with endophytes exhibited less membrane damage than plants under drought stress. The literature has reports of comparable outcomes when endophytes were cultured on wheat and tomatoes [44]. These findings highlight the potential of using fungal endophytes as a biological way to improve plants' resistance to drought. The potential of endophytes to lessen the effects of oxidative stress and stabilize membranes under water deficit conditions are essential to preserving plant health and efficiency in arid regions. Future studies have to concentrate on comprehending the molecular processes behind endophyte-mediated stress tolerance and exploring the application of these findings in various crops and environmental conditions.

Antioxidant enzymes

Different variations in the activity of antioxidant enzymes were generated by drought stress. Table 5 illustrates that at the highest level of drought stress, plants exhibited reduced CAT and SOD activities but increased APX and GR activities. There was a favorable association between increased drought level and APX and GR activity. Activities of GR and APX were reduced by the worsening drought. However, CAT, SOD, APX, POD, and GR activities increased in the drought-stressed plants treated with fungal endophytes. All fungal endophyte treatments colonized the plants even at the highest level of drought stress. The most effective treatment increased SOD activity in T. vulgaris leaves (89.99%) (Fusarium sp.) by less than half (CAT activity increased by 183.75%; C. Puyae and C. australiensis) (Table 5). According to Table 5, it caused C. Puyae and C. australiensis to enhance APX activity by 39.22%, GR activity by 283.02% (by C. australiensis) (Table 5) and POD activity by 251.09% (by C. australiensis) (Table 5), under drought stress conditions at 50% FC, in contrast to the uncolonized plants. A high rate of antioxidant enzyme activity can lessen the negative effects of drought stress [45]. Fungal endophytes play a vital part in improving plant resilience under drought conditions by augmenting intracellular scavenging enzymes like CAT and SOD [46]. antioxidant enzymes, like CAT facilitate the conversion of H₂O₂ into water, thereby reducing oxidative stress [47]. M. oleifera's activation of genes encoding antioxidant enzymes following endophyte inoculation underscores their role in regulating drought stress tolerance [48]. Studies have reported the enzymes GR, POD, SOD, APX, and CAT being regulated activated in response to stress conditions [49]. Through the transformation of superoxide radicals, SOD serves as the main defense against ROS. [50], while CATs protect cells from oxidative damage [51]. In plants inoculated with fungal endophytes, CAT and SOD activity significantly increases under drought stress, highlighting the protective role of endophytes in scavenging ROS (T. vulgaris plants) [52]. Glutathione levels under drought stress are significantly greater in endophyte-inoculated plants than in controls, which is in line with earlier research under cadmium exposure [53]. The application of C. australiensis enhances GR activity, and combined with C. puyae, maximizes APX activity to counteract the toxic effects of drought stress [52]. Fungal endophytes like F. equiseti upregulate various antioxidant genes, including those encoding ascorbate and glutathione, thereby increasing plant resilience to oxidative stress [54]. The findings underscore Fungal endophytes' potential as biotechnological tools to improve plant antioxidant capacity and mitigate oxidative stress under adverse environmental conditions. It is important for future studies to clarify the molecular processes that underlie these interactions and how they work in various plant species and environmental conditions.

Correlation analysis

Hierarchical clustering analysis (HCA)

HCA is A normalized heatmap matrix was constructed in order to evaluate the general responses of morphological, physiological, biochemical, and antioxidant specifics of T. vulgaris under drought stress and fungal endophytes. in Fig. 3, Changes in the various attributes are visible of T. vulgaris under drought levels. Based on the relative responses of parameters to treatment and drought severity level, two significant clusters were found. The plants in the first cluster (I), which contained both control and fungal endophyte-treated plants, had low weight loss and high to extremely high antioxidant levels. Plants in the second group (II) had low to extremely low morphological and physiological features; these plants were among those enduring drought. fungal endophytes + drought treatment revealed less drought, higher APX, SOD and NL than other treatments. Treatments such as F₁D₀, F₃D₀, and F₂ + F₃D₀ are clustered close to each other, indicating they have similar effects on various traits. Treatments like F₂ + F₃D₂ and F₃D₁ are also clustered together, suggesting similar impacts on various traits. Traits at the top of the heatmap are separated into four groups (A, B, C, and D). which are marked with different colors. These groups represent different categories of traits: Group A (Blue): Morphological traits (LF, LD, SF, SD, RF, RD, SL, RL, RW, NS, NL, TD), Group B (Green): Physiological traits (Chl a, Chl b, Chl T, Caro), Group C (Purple): Antioxidant traits (SOD, APX, POD, GR, CAT), Group D (Red): Biochemical traits (MDA). Traits that are closer together show similar patterns of treatment effects. For example: Traits related to leaf fresh and dry weight (LF, LD) are close to each other, indicating similar treatment effects on these traits. Traits related to biochemical enzyme activities (SOD, APX, POD) are also close to each other, suggesting these traits are similarly affected by treatments. Drought Treatments (D) are generally shown in areas with red colors on the heatmap, indicating stronger impacts on certain traits. For instance, drought treatments may have a more significant effect on morphological traits like leaf and stem fresh and dry weight. Fungal and Control Treatments (N) might have lesser effects compared to drought treatments, often appearing in areas with blue or less red colors. For example, fungal and control treatments may have less impact on traits related to antioxidant enzyme activities. The clustering of treatments and traits shows which treatments have similar effects on various traits and which traits are similarly affected by different treatments. The trait groups (A, B, C, D) help understand which categories of traits are similarly affected by the treatments.

Pearson’s correlation analysis

The purpose of this analysis was to examine the association between antioxidant activity under stress and normal conditions, as well as growth and physyological and biochemical markers. These elements were mainly concentrated on and put together as a single unit for the creation of heatmaps (Fig. 4A,B). The circle represents the Pearson correlation coefficient between two parameters. The color and size of the circle indicate the magnitude and direction of the correlation: Dark Blue Color: High positive correlation, Red Color: Negative correlation, Smaller Circles: Correlation close to zero, Many growth parameters such as LF, LD, SF, SD, RF, RD, SL, RL, RW, NS, NL, and TD have a high positive correlation with each other. This indicates that these parameters change in tandem with each other. chl a and chl b also have a high positive correlation with each other, indicating their interdependence. Some parameters like SOD, APX, POD, GR, CAT, and MDA have a negative correlation with other parameters like LF, LD, SF, SD, etc. This indicates that as one of these parameters increases, the other decreases. Based on the analysis of the correlation matrix and the resulting findings, Growth-related parameters of the plant show a high positive correlation with each other, indicating mutual positive effects among them. Antioxidant activities like as SOD, APX, POD, GR, and CAT have a negative correlation with some of the growth parameters. This could indicate the protective effects of these activities under stress conditions.

Principle component analysis (PCA)

To learn more about the relationship and impact of various treatments, PCA was used (Fig. 5A, B). Response variables for morphological, physiological, biochemical, and antioxidant activities were used to build the PCA. These results indicated that Many growth parameters such as LF, LD, SF, SD, RF, RD, SL, RL, RW, NS, NL, and TD have a high positive correlation with each other. This indicates that these parameters change in tandem with each other. Additionally, chl a, chl b also show a high positive correlation, suggesting their interdependence. Some parameters like SOD, APX, POD, GR, CAT, and MDA have a negative correlation with other parameters like LF, LD, SF, SD, etc. This indicates that as one of these parameters increases, the other decreases. Based on the analysis of the correlation matrix and the resulting findings, The growth-related parameters of the plant show a high positive correlation with each other, indicating mutual positive effects among them. Antioxidant activities like as SOD, APX, POD, GR, and CAT have a negative correlation with some of the growth parameters. This could indicate the protective effects of these activities under stress conditions. The biplot visualizes the relationships between the active variables (red points) and active observations (blue points). It is constructed using the first two principal components (F1 and F2), which together explain 73.00% of the total variance (51.00% by F1 and 22.00% by F2). Growth Parameters: Observations ND1, ND2, and ND3 (drought conditions) are positioned away from the origin on the negative side of F1, indicating lower values for growth parameters compared to other treatments. The parameters SOD, APX, POD, GR, and CAT are clustered together, reflecting their similar behavior and high correlation under different treatments. chl a, chl b and chl t are positioned closely, showing their strong interdependence. MDA (Malondialdehyde) is positioned far from the cluster of growth parameters, indicating its negative correlation with growth. The biplot effectively demonstrates the relationships and groupings among different variables and treatments, highlighting how different factors impact plant growth and stress responses.

Here, we evaluated the possibility of introducing three fungal endophytes into T. vulgaris plants through inoculation. Individually and in combination, treatments with Fusarium sp., C. puyae, and C. australiensis considerably (P < 0.001) enhanced the performance of plants. Under drought stress, their treatments enhanced T. vulgaris plant growth. When compared to drought stress alone, the inoculation of this fungal endophyte greatly enhanced the following aspects of plant growth: length of shoot and root, leaves and root biomass, shoot diameter, root breadth, number of leaves and branches, concentrations of chlorophyll and carotenoid, and antioxidant activity. This demonstrates how fungal endophytes acted as a regulator to lessen the effects of drought stress. The C. australiensis exhibited the maximum degree of plant growth and resilience against the effects of drought. our results suggest that fungal endophytes exhibit different responses to drought stress, and utilizing tolerant inoculants could serve as a feasible and efficient approach for enhancing T. vulgaris growth in water-deficient situations. The high drought tolerance of T. vulgaris inoculated with Fusarium sp., C. puyae, and C. australiensis was discovered to produce larger levels of photosynthetic pigments than the uninoculated control, raising photosynthetic activity and biomass accumulation. Additionally, it was discovered that treating T. vulgaris with a combination of fungal endophytes was more beneficial than inoculating the plant with a single fungal endophyte to promote early development in the presence of water restrictions. Both directly and indirectly -via redox antioxidants like glutathione- ROS leave cells through pathways like CAT, APX, SOD, and POD. Isolated endophytes showed the ability to develop effectively under drought stress without abandoning their potent cellular machinery for antioxidant production, as revealed by the activities of GR, POD, APX, SOD, and CAT. The results also suggest that fungal endophytes may be able to lessen the harmful effects of ROS by managing antioxidants and related enzymes. More plant growth-promoting benefits could be enhanced by fungal endophyte inoculation, comparable to the well-documented effects of osmoprotectants and PGPR in agricultural plants. According to our research, horticulturists can use fungal endophytes as an environmentally safe method to lessen T. vulgaris's harmful impacts from drought stress. The findings show a notable improvement in drought tolerance, reduction in lipid peroxidation levels, and enhancement of antioxidant enzyme activities with the use of these endophytic fungi. This research emphasizes the significance of drought-resistant fungal endophytes and their combinations, showcasing how collaborative fungal interactions can be utilized to create a cost-effective biofertilizer for farmers in arid regions. Consequently, conducting field trials to explore the potential utilization of local biofertilizers in dryland agricultural setups is recommended for future investigations.

Collection of Plant Samples

In this study, plants from the Lamiaceae family were collected on October 31, 2022, from Mount Geno in Hormozgan Province, Iran (27.4173° N, 56.1422° E), at an elevation of 2347 meters above sea level. The identification of these plants was carried out by Mohammad Amin Soltani Pour, a plant taxonomist at the Agricultural and Natural Resources Research Center of Hormozgan Province. We also used resources associated with him [55] to confirm the identifications. The plants were matched with herbarium numbers (HN) available at the research center, including: Menta mozaffarianii (HN: 6331), Teucrium pollium (HN: 6307), Zataria multiflora (HN: 6297), Lavandula stricta (HN: 6312), Zhumeria majdae (HN: 6320), Otostegia persica (HN :6323), Salvia sharifii (HN: 6324), Teucrium stockisanum (HN: 4064), and Teucrium orientale (HN: 6295).

Endophyte Material

Isolation, Purification, Identification, and Screening of Fungal Endophytes

Following sterilization, split into smaller sections, and disinfection, plant samples were grown in Potato Dextrose Agar (PDA) media. After growing for four to six weeks, fungal endophytes were purified using the hyphal tip method and then placed in a fresh PDA culture to continue growing [56]. Fungal isolates were evaluated using microscopic slides stained with lactophenol according to the identification key established by Barnett and Hunter (1997) [57]. and seen under a light microscope. Following the extraction of genomic DNA, PCR amplification was carried out. The use of agarose gel electrophoresis allowed for the evaluation of DNA quality. Purified fungal cultures were then based in test tubes with PDA and kept at 4 degrees Celsius until needed again. In this experiment, the optical density (OD) fungus suspension was modified to a concentration of 10⁶ cells per milliliter. 10 ml of fungal suspension were sprayed over the leaves of every T.vulgaris plant. Finally, a test evaluating fungal isolates' resistance to drought was carried out under laboratory conditions (Fig. 1b) [58].

Plant Material

In the present study, Fusarium sp. isolated from the root of Zhomeria majdae, C. puyae isolated from the shoot of Teucrium stocksianum and C. australiensis isolated from the root of Salvia mirzayanii, exhibited the highest drought tolerance levels and were selected as treatment modalities for this research. To conduct the experiment, T. vulgaris seeds sourced from Hatam Agricultural Growth Company in Shiraz, Iran, were utilized. To initiate the experiment, T. vulgaris seeds were first immersed in 70% ethanol for 30 seconds, sterilized with 0.5% sodium hypochlorite for 90 seconds, and then thoroughly rinsed twice with distilled water. For better contact with endophytes, 1% carboxymethyl cellulose was used, and the inoculated seeds were placed on a shaker for 6 hours. Control seeds were shaken in distilled water containing 20% Tween for 6 hours. Following this inoculation process, seed sowing was conducted in germination trays. After 2 months, the seedlings were transferred to the main pots. Standard techniques were employed to measure the potting soil's physical and chemical characteristics (Table 1). seeds in a vase with a diameter of 21 cm and a depth of 22 cm that was loaded with autoclaved substrates (0.1 MPa, 121°C, 1 hour). Sand, cocopeat, and peat moss (v/v/v; 2:1:1). This treatments for drought stress were put into place. After 4 months, once the plants had sufficiently grown and developed four lateral branches, they were inoculated with endophytes over a one-month period, administered in two stages. This secondary inoculation was conducted to evaluate the drought resistance of control plants compared to treated plants under drought stress conditions. After the final inoculation stage, leaf samples were randomly selected from each treatment group of T. vulgaris plants inoculated with fungal endophytes. Following sterilization, the leaf samples were individually cultured on PDA media to verify the establishment of the target endophytes within the plant tissue. The successful growth of the previously inoculated fungal endophytes on the PDA culture media served as confirmation of their establishment within the plants. Following confirmation of the presence of fungal endophytes in the host plant, drought stress treatments were applied for a duration of 3 months. The experimental treatments included 4 irrigation levels (100, 75, 50, and 25% of field capacity) and different levels of endophytes. The experimental design was a factorial arrangement in a completely randomized design (CRD) with 3 replications. The plants were cultivated in a greenhouse under ambient light conditions, with a photosynthetically active radiation (PAR) ranging from Around 700–950 µmol m^− 2 s^− 1. The greenhouse maintained a 12-hour photoperiod, with the temperature regulated within the range of 25–28°C, and relative humidity controlled between 65–70%.

Endophytic fungus

To plant inoculation, three isolates out of 44 with the highest tolerance to drought test were selected. The isolates were noted as Fusarium sp. (Accession number of OP743376), C. Puyae (Accession number of OP811006) and C. australiensis (Accession number of OP811007) in NCBI gene bank. fungal endophytes were grown in the nutritional media with the addition of PEG 6000 and their responses to environmental drought have been investigated. The grew on -3 bar (osmotic potential) medium. The capacity of a specific type of fungus to survive in extremely dry conditions is crucial for plants to adapt to stressful conditions and can provide insight into the survival strategies employed by endophytes in extremely dry natural settings. These endophytes were chosen because of their ability to withstand soil stress and because they can continue to benefit plants in comparable the environment.

Fungal inoculation

Three duplicates of each treatment were used in the plant inoculation experiment. (1) Fusarium sp. (F₁), (2) C.Puyae (F₂), (3) C. australiensis (F₃), (4) F₁ + F₂, (5) F₁ + F₃, (6) F₂ + F₃, (with cell density of 1x10⁶ CFUml^− 1) were used to six-months-old T. vulgaris. Additionally, an equal volume of Distilled water was employed as a control. Soil drenching and applying foliar spray were used to inoculate the plants with suspensions of both individual and combination fungal endophytes. 10 ml of each microorganism's suspension were sprayed over the leaves of every T. vulgaris plant. Five ml of the suspensions were applied to the soil around the plant's crown in order to soak it. Distilled water that had been sterilized was used to irrigate the plants

Drought stress treatments

Following the establishment of endophytes in the plants, four irrigation levels (100, 75, 50, and 25% FC) were applied to the plants after a month. Using the weight fraction, the soil water content (SWC) was calculated as follows: The weight fraction was used to calculate the soil water content (SWC) as follows:

𝑆𝑊𝐶 (%) = [𝐹𝑊 – 𝐷𝑊/ 𝐷𝑊] × 100

where FW was the soil's fresh weight from the interior of each pot and DW was determined by the soil's weight loss following three days of oven drying at 75°C [59].

Plant harvesting

Three months after Drought stress, the impact of fungal endophytes on drought resistance was assessed by the use of antioxidant, biochemical, and morphological traits. All plants were promptly harvested once the stress period ended, and Healthy leaves were frozen in liquid nitrogen and kept at a temperature of -80°C. During harvest, growth parameters, root length, root width, shoot length, trunk width, leaf and branch number were documented.

Plant growth measurement

Measurements included root length, root width, leaf and branch count, fresh/dry weight of leaves, fresh/dry weight of roots, shot length, and trunk width. Following 48 hours of oven drying at 70°C until a fixed weight was reached, the dry weight (DW) of the leaves, shoots, and roots was measured.

Calculating photosynthetic pigments

By extracting 0.2 g of leaf material in 10 ml of acetone (80%), Carotenoids, Chlorophyll a, Chlorophyll b, and total chlorophyll were measured [60].

Malondialdehyde (MDA)

The method outlined by Hodges et al. (1999) [61], was employed to determine the MDA concentration in leaves. To do this, 200 mg of leaf powder and 2.5 ml of 0.1% TCA (trichloroacetic acid) were combined, and the mixture was centrifuged at 10,000 × g for 15 minutes at 4°C. After that, 1 ml of 0.5% thiobarbituric acid (TBA) was added to the supernatant and 20% trichloroacetic acid (TCA). The blend mentioned above was cooked for approximately 20 minutes at 90°C in a bath of water. Following cooling in an ice bath, the mixture centrifuged at 15,000 × g for 10 minutes. At 532 nm, the absorbance was computed.

Antioxidant enzyme activity

200 mg of leaf powder per antioxidant enzyme was homogenized in an extraction buffer (Potassium phosphate buffer, 50 mM) with 1% (w/v) poly vinyl pyrrolidone (PVP) at pH 7.5. After mixing, it was centrifuged for 15 minutes at 4°C at 10,000 × g. The supernatant was utilized for the antioxidant enzyme assay and kept at -20°C.

Superoxide Dismutase (SOD)

According to Becana et al. (1986) [62], superoxide dismutase (SOD) activity was evaluated based on its capacity to prevent nitro blue tetrazolium (NBT) from being photo reduced. 50 microliter of enzyme extract and 50 millimeters of pH-neutral potassium phosphate buffer (7.5), 14.3 mM methionine, 82.5 mM NBT, 0.1 mM EDTA, and 2.2 mM riboflavin were all included in the combination of reactions (1 ml). Fifteen W fluorescent lights were used to illuminate the process. Ten minutes later, the reaction came to an end. At 560 nm, the absorbance was measured.

Catalase (CAT)

Measurements of catalase (CAT) were made in accordance with Chance and Maehly (1955) [17], 100µl of enzyme extract, 4.4 mM H₂O₂, and 50 mM potassium phosphate buffer (pH 7.0) made up the reaction mixture (1 ml). At 240 nm, the absorbance was estimated (ε = 39.4 mM-1 cm − 1).

Ascorbate peroxidase (APX)

The activity of ascorbate peroxidase (APX,) was measured using the Asada (1984) [63] protocol. An enzyme extract of 33µl, 0.17 mM ascorbate, and Potassium phosphate buffer, 50 mM (pH 7.0) made up the combination of reactions (1.0 ml). The addition of 5 mM H2O2 started the process. For three minutes, the absorbance was determined at 290 nm (ε = 2.8 mM^− 1 cm^− 1).

Peroxidase (POD)

For POD, a 3 ml mixture was made in 50 mM potassium phosphate buffer pH 7.0 with 0.5 mM EDTA, 30µl of enzyme extract, and 2970µl of guaiacol (45 mM) and H₂O₂ (100 Mm). The oxidation of guaiacol when H₂O₂ is present (extinction coefficient of 26.6 mM-1 cm-1) at 470 nm during intervals of two minutes was used to measure POD activity [63].

Glutathione Reductase (GR)

Glutathione reductase (GR) activity was measured by applying the Smith et al. (1989) [64]. technique. The reaction mixture (1.0 ml) included 0.75 mM DTNB (5, 5′dithiobis − 2nitrobenzoic acid), 1 mM GSSG, 100µl of crude enzyme extract, and 50 mM potassium phosphate buffer (pH 7.5). The reaction was started by adding 0.1 mM NADPH. At 412 nm (ε = 14.15 mM − 1cm − 1), the increase in absorbance resulting from the production of TNB (5-thio-2-nitrobenzoic acid) was measured.

Statistical analyses

The received sequences were edited using SeqMan software and compared to other sequences available in the NCBI gene bank using BLAST. Data analysis was performed using SAS software (version 9.4), and mean comparisons were evaluated using the LSD test at at the level of P < 0.05. The analysis, conducted using XLSTAT software and the Shapiro-Wilk test, revealed a P-value ≥ 0.05, indicating that our data adhere to a normal distribution. Consequently, parametric tests were employed for data analysis (Shapiro and Wilk, 1965). Cluster analysis and The PCA was conducted using XLSTAT program, version 2020 (www.xlstat. com, Addinsoft SARL). Heatmap and Correlation Analysis was conducted using SRPLOT (on line) [65].

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

Correspondence and requests for materials should be addressed to D.S

Competing interests

The authors declare no competing interests.

Funding

The current research has received no funding from agencies in the public, commercial, or not-for-profit sectors.

Acknowledgments

Author Contributions

Methodology, A.K; Software, A.K; Validation, A.K., D.S; Formal analysis, A.K.; Investigation, D.S, N.A. and A.B.; Resources, A.K., D.S; Writing-original draft, A.K; Writing-review & editing, A.K and D.S.; Supervision, D.S.; Project administration, A.K., DS; Data curation, A.K., DS; Conceptualization, A.K, D.S and N.A. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Lamiacea family-Derived Endophytic fungi: Induced Tolerance to drought Stress in Thymus vulgaris plants

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Abstract

Figures

Introduction

Results and Discussion

Growth parameters

Chlorophyll and Carotenoid

MDA content

Antioxidant enzymes

Correlation analysis

Hierarchical clustering analysis (HCA)

Pearson’s correlation analysis

Principle component analysis (PCA)

Conclusion

Methods

Collection of Plant Samples

Endophyte Material

Isolation, Purification, Identification, and Screening of Fungal Endophytes

Plant Material

Endophytic fungus

Fungal inoculation

Drought stress treatments

𝑆𝑊𝐶 (%) = [𝐹𝑊 – 𝐷𝑊/ 𝐷𝑊] × 100

Plant harvesting

Plant growth measurement

Calculating photosynthetic pigments

Malondialdehyde (MDA)

Antioxidant enzyme activity

Superoxide Dismutase (SOD)

Catalase (CAT)

Ascorbate peroxidase (APX)

Peroxidase (POD)

Glutathione Reductase (GR)

Statistical analyses

Declarations

References

Additional Declarations

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