Mitigation of Early Blight in Tomato Plant: Evaluating Trichoderma harzianum, Cassia fistula and Azadirachta indica leaves powder

doi:10.21203/rs.3.rs-4534571/v1

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Mitigation of Early Blight in Tomato Plant: Evaluating Trichoderma harzianum, Cassia fistula and Azadirachta indica leaves powder

https://doi.org/10.21203/rs.3.rs-4534571/v1

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Early blight, caused by the fungus Alternaria solani, is a significant challenge in tomato cultivation, often leading to yield losses. In this study, we investigated the potential of three natural substances: Trichoderma harzianum (spore suspension), Cassia fistula and Azadirachta indica (leaves powder) in mitigating early blight. These substances were applied individually and in combination with all of three substances as treatments to evaluate their efficacy in disease suppression in tomato seedlings. The experiment was conducted in a Completely Randomized Design with three replications. After 15 days of various growth parameters like root and shoot length, fresh and dry weight were studied. Furthermore, the biochemical (Catalase and Peroxidase activities) and physiological (Total protein content and reducing sugar) attributes of tomato plants were also investigated. The results revealed that the growth parameters of tomato seedlings were significantly declined by 70 to 80% when infested by early blight disease. However, soil amendments with T. harzianum + C. fistula nullify the effect of the pathogen and increased the plant growth. Furthermore, the physiological and biochemical traits exhibited more production (up to 90%) and less activity (~ 20 to 30%), respectively in plants when plants were grown in T. harzianum + C. fistula in comparison with control. The trend of better growth in plants was T. Harzianum + C. fistula > T. harzianum > C. fistula >. Therefore, the soil amendment with T. Harzianum + C. fistula in pathogen-infested soil could be implemented to combat the early blight of disease. The results indicated that bio-agents such as C, fistula and A, indica in combination with Trichoderma can be used in antifungal products. Overall, this study highlights the promising role of natural substances in integrated pest management strategies for sustainable tomato cultivation. Further research is warranted to elucidate the underlying mechanisms of action and optimize application protocols for maximizing efficacy while minimizing environmental impacts.

Early blight disease

bio fungicides

Trichioderma

Neem leaves

Tomato (Solanum lycopersicum. L), also known as Nightshades, belongs to the Solanaceae family, having 3000 species all over the world. (1). It is the second most significant vegetable, a widely cultivated crop with a substantial per capita consumption, and responsible for generating 14% of the global vegetable production (2). Over the past few decades, the worldwide annual tomato production has experienced a consistent growth, as reported by the FAO. The estimated figure stands at approximately 123 million tons, cultivated across a total production area of about 4.5 million hectares. Moreover, the global production of fresh tomatoes surpasses a staggering 159 million tons annually (3).

Tomatoes are known for good source of vitamins, minerals, and bioactive substances known as secondary metabolites, the concentrations of which are linked to reducing the risk of developing chronic degenerative diseases in people, including cancer, cardiovascular disease (CVD), and neurological illnesses (4). Tomatoes can help treat a variety of illnesses, due to their high concentrations of various natural antioxidant chemicals, including carotenoids (carotenoids and lycopene), ascorbic acid (vitamin C), tocopherol (vitamin E), and bioactive phenolic compounds like quercetin, kaempferol, naringenin, and lutein (5). Lycopene, which is abundant in tomatoes and tomato-based products, is primarily responsible for the sparkling red color of many fruits and vegetables. Lycopene in tomatoes plays a vital role in delaying cell damage, preventing various stomach-related diseases, gastrointestinal issues, and prostate cancers (6).

There are various biotic and a-biotic factors, which are responsible in decaling the plant yield. Among all, biotic agents, Alternaria solani a lethal fungal pathogen, is responsible of causing Early Blight in tomato (7). Early blight is a most concerning disease, due to which a lion's share of the crop is subjected to garbage. This pathogen belongs to the Ascomycota family, characteristic of producing large spores which infects the stem, and fruits of the tomato plant (8). After infection, A. solani causes lesions in leaves and stem, reduced overall plant growth and fruit size (9). To control this fungus various fungicides are being used by farmers, but often, pathogens develops resistance towards these chemicals. Moreover, these fungicides are entering into our food chain via the process of bio-magnification and bio-accumulation (10). By entering into the food chain, not only plants, animals and human health is also at risk.

Adopting biodegradable and environmental friendly approaches is a need of a hour, as bio-agents and plant extracts can be consider a new rays of hope, due to eco-friendly nature and can be used an alternative measure to control fungal diseases Bio agents like the leaves of Cassia fistula (Amaltas) and Azadirachta (Neem) are well known for their medicinal benefits and used by people from centuries to cure various diseases. (11). Neem is used to cure cardiac issues and acts as an antifungal, antibacterial, anti-allergic, anti-demonic, anti-inflammatory, antipyorrheic, and anti-scabic (12). Whereas C. fistula leaves contain different types of antioxidants which make them an important agent against various plant pathogens. In addition, Cassia Leaves shows antioxidant properties due to the presence of triterpenes, Phenolic and flavonoid contents (13).

Apart from plants metabolites, antagonistic fungi like Trichoderma harzianum has studied in great deal of studies which indicated that along with plants powder, Trichoderma has improved the plant growth and yield. Trichoderma produces a wide range of secondary metabolites such as terpenoids, alkaloids and melatonin (14). Melatonin is a low molecular weight IAA-like structure that acts as a growth regulator and help plants to survive and shows better growth even in extreme stress environment (15). On the other hand, Trichoderma is a well-known fungus to forms mutual beneficial association with plants and therefore enhance over all plant growth by causing resistance against fungus and bacterial pathogen (16).

In the present study, plants leave powder and Trichoderma harzianum are used to control early blight disease in tomatoes. Such type of biodegradable approaches seems to be a ray of hope in mitigation of pathogen stress in tomato plant.

The experiment was carried out at botanical garden, School of botany, Minahj University Lahore, during the Rabi season. Field experiment was laid out in completely randomized design (CRD) with seven treatments having three replications.

Isolation and Identification of Fungus

Both fungus culture A. solani and T. harzianum (FCBP- PTF-831, 1277) was acquired from the (First Fungal Culture Bank of Pakistan, Institute of Agriculture Sciences, University of Punjab, Lahore. Sub culturing was done by growing fungus in PDA media, incubated at 25 ºC for 5–7 days. After7 days, pure fungal cultures were further characterized (17).

Suspension Preparation of T. harzianum

About 1 mL of sterilized autoclaved distilled water was added to the fully grown fresh mother culture of T. harzianum scraped with a spade to produce a slurry and then transferred to 99 mL of distilled water to make a suspension. About 1000 µL of fungal suspension was taken in a sterile Eppendorf tube and stored at room temperature.

Plant material preparation

Two medicinal plants, i.e., C. fistula and A. indica leaves, were collected from the vicinity of Minhaj University, Township Lahore. The leaves were washed up with double distilled water and fine powder was made. For Pot experiment tomato seedlings were procured from the Nursery located in Model Town Link Road, Lahore.

Sandy loam soil was sterilized with formalin covered with plastic sheets, and kept for seven days under sterilization. After seven days, the plastic cover was removed and left for seven days to remove the formalin fumes from the soil. After that, the sterilized soil was filled in a plastic pot. The seedlings of tomato (about 2 seedlings) were planted in the soil. Furthermore, the experiment was carried out with seven different treatments i.e., -ive control, positive control, T. harzianum (TH) + A. Solani (AS), C. fistula (CF) + AS, A. indica (AI) + AS, TH + AI + AS and TH + CF + AS. The whole of the experiment was carried out in triplicates with completely randomized design. A total of 18 pots were used in this experiment. Each replicate was treated with a different combination of treatments. Table 1 describing the work plan.

Table 1

The whole experiment was divided into 5 treatments. Plants were plucked after 45 days before flowering stage.
Sr. no	Treatments	Treatments detail	Application
1	T₀	Control (untreated)	Distilled water spray
2	T₁	Fungus (Alternaria solani)	Soil amendment
3	T₂	Cassia fistula + Alternaria solani	Soil amendment
4	T₃	Azadirachta indica + Alternaria solani	Soil amendment
5	T₄	Trichoderma harzianum + Alternaria solani	Soil amendment
6	T₅	Trichoderma harzianum + Cassia fistula + Alternaria solani	Soil amendment

Tolerance index

After 25 days of sowing, the tomato plants were analyzed for pathogen symptoms (percentage chlorosis or yellowing in foliage), disease severity index, plant mortality, and tolerance index (T _index).

G_i = \(\frac{\text{G}\text{x}}{\text{G}\text{m}\text{a}\text{x}}\)

T _index = \(\frac{\sum \left(\frac{Gx}{Gmax}\right)}{total number of parameters}\)

Where, G_i =normalized growth parameter; Gx = individual growth or yield parameter; Gmax = maximum value; T_index = Tolerance index.

Physiobiochemical assays

Physiological attributes like total chlorophyll content (TCC), Catalase, reducing sugar (RS), and total protein content (TPP) and Peroxidase activities were assays tomato plant after 25 days.

Total Chlorophyll Content (TCC)

Total chlorophyll content was assayed by using (Lichtenthaler, 1987) method 500 mg of fresh leaf material was crushed in 10 mL of 80% ethanol. Subsequently, the mixture was subjected to centrifugation at 2000 rpm for approximately 20 minutes. Following this, the absorbance was measured at wavelengths of 470 nm, 645 nm, and 663 nm

Total Protein Content (TPC)

To determine the total protein content, 0.1 mL of fresh leaf material was blended in approximately 10 mL of phosphate buffer. Following this, the mixture underwent centrifugation at 2000 rpm for 20 minutes (17). For the next step, a test tube was used, where 0.1 mL of the supernatant, 0.9 mL of distilled water, and 1 mL of reagent C were combined and shaken for a duration of 10 minutes. Subsequently, 0.1 mL of reagent D was introduced into the same tube, and the mixture was left to incubate for 30 minutes. After this incubation period, the absorbance was measured at 660 nm. (18) These same procedures were replicated for all other treatments, ranging from T0 to T7.

Reducing Sugar (RS)

To measure the concentration of reducing sugar, 1 mL of the plant sample was first homogenized in approximately 10 mL of 80% ethanol, followed by centrifugation at 2000 rpm for approximately 20 minutes. Subsequently, 1 mL of the ethanol extract was mixed with 1 mL of 5% Folin phenol and 5 mL of 96% H₂SO₄. The mixture was thoroughly agitated at room temperature for 20 minutes, after which the absorbance was measured at 490 nm (19)

Catalase activity (CAT)

To measure catalase content, 1 mL of the plant sample was homogenized with 10 mL of 0.1 m phosphate buffer and then subjected to centrifugation at 4°C, at 10,000 rpm for 10 minutes. Following this, 1 mL of the supernatant was collected, and to it, 1 mL of 0.01 m H₂O₂ and 1 mL of 0.1 m phosphate buffer were added. The mixture was incubated for 5 minutes at 20°C. Subsequently, 10 mL of 1% H₂SO₄ was introduced, and the solution was titrated against 0.005 N KMnO₄ until the appearance of a pink color signified the endpoint of the titration.

Peroxidase Detection (POD)

To assess peroxidase levels, 0.5 mL of the plant material sample was homogenized with 10 mL of 0.1 m phosphate buffer in a test tube and then subjected to centrifugation at 4°C, at 10,000 rpm for 10 minutes. Following this, 0.5 mL of the supernatant was collected, and 1 mL of phosphate buffer, 1 mL of pyrogallol, and 1 mL of 0.01 m H₂O₂ were added. The mixture was then incubated at 25°C for 5 minutes, and the absorbance at 595 nm was measured by using a method designed by (20).

GCMS Analysis

Gas Chromatography machine model 7890B (Agilent) and Mass spectroscopy was 5977 (Agilent) used. The column used was DB 5 MS (30 m × 0.25 µm × 0.25 µm) Injection volume was 1 µl and carrier gas was helium, initial temperature was 80 ºC and then raised 10 ºC per minute up to 300 ºC. Chemical compounds were distinguished by comparing their spectra against a reference library and then sorted in increasing order based on their retention times and retention indices. Their respective abundance was quantified by assessing the peak areas.

Statistical Analysis

Data was analyzed by using analysis of variance and the difference between the treatments was accessed by employing the LSD test at 5% probability. By using Origin Pro, Correlation and Principal Component Analysis PC₁ and PC₂ were also determined.

Morphological Alternations

The plant grown under the stress of A. solani exhibited limited growth in tomato plants. However, growth of the tested plant has been improved when the soil was amended with T. harzianum, leaves powder of Cassia fistula and A. indica. Moreover, the disease did not appear any obvious appearance (Fig. 1).

Growth Parameters

Shoot parameter

The results revealed that the shoot length of tomato plants was improved under the application of T. harzianum and plant leaves powder. The findings revealed a significant (P ≤ 0.05) reduction in the shoot length of tomato plants in positive control (A. solani) by 52% as compared to negative control. However, the shoot length of examined plant was significantly increased by -75% (T. harzianum + A. solani), -57% (C. fistula + A. solani), -20% (A. indica + A. solani), -87% (T. harzianum + A. indica + A. solani) and − 66% (T. harzianum + C. fistula + A. solani) respectively as compared to positive control (Fig. 2).

The results indicated a significant (P ≤ 0.05) reduction in shoot fresh weight of tomato plants in positive control by 90% (A. solani) as compared to negative control. However, the shoot fresh weight of the examined plant was significantly increased by -32% (T. harzianum + A. solani), -172% (C. fistula + A. solani), -134% (A. indica + A. solani), -103% (T. harzianum + A. indica + A. solani) and − 138% (T. harzianum + C. fistula + A. solani) respectively as compared to positive control (Fig. 2).

The results exhibited that a significant (P ≤ 0.05) reduction in shoot dry weight of tomato plants in positive control by 76% (A. solani) as compared to negative control. However, the shoot dry weight of examined plant was significantly increased by -529% (T. harzianum + A. solani)), -69% (C. fistula + A. solani), 0% (A. indica + A. solani), -159% (T. harzianum + A. indica + A. solani) and − 94% (T. harzianum + C. fistula + A. solani) respectively as compared to positive control (Fig. 2)

Root parameters

The results indicated the significant (P ≤ 0.05) reduction was observed in root length of tomato plant in positive control by 47.27% (A. solani) as compared to negative control. However, the root length of examined plant was significantly increased by -25% (T. harzianum + A. solani), -40% (C. fistula + A. solani), -40% (A. indica + A. solani), -36% (T. harzianum + A. indica + A. solani) and − 75% (T. harzianum + C. fistula + A. solani) respectively as compared to positive control (Fig. 3).

The results indicated a significant (P ≤ 0.05) reduction in root fresh weight of tomato plant in positive control by 75% (A. solani) as compared to negative control. However, the root fresh weight of examined plant was significantly increased by -6% (T. harzianum + A. solani), 35% (C. fistula + A. solani), -132% (A. indica + A. solani), -230% (T. harzianum + A. indica + A. solani) and − 213% (T. harzianum + C. fistula + A. solani) respectively as compared to positive control (Fig. 3).

The results indicated a significant (P ≤ 0.05) reduction in root dry weight of tomato plants in positive control by 52% (A. solani) as compared to negative control. However, the root fresh weight of examined plant was significantly increased by 85% (T. harzianum + A. solani), 88% (C. fistula + A. solani), 46% (A. indica + A. solani), -117% (T. harzianum + A. indica + A. solani) and − 88% (T. harzianum + C. fistula + A. solani) respectively as compared to positive control (Fig. 3).

Physiological Parameters

It was revealed that the impact of T. harzianum and different leaves powder of C. fistula and A. indica on total chlorophyll content of tomato plant grown under the stress of A. solani was significantly (P ≤ 0.05) declined up to 76.11% (A. solani) over negative control. However, the total chlorophyll content of tested plant was significantly increased by -47% (T. harzianum + A. solani), -26% (C. fistula + A. solani), -72% (A. indica + A. solani), -107% (T. harzianum + A. indica + A. solani) and − 130% (T. harzianum + C. fistula + A. solani) respectively as compared to positive control (Fig. 4).

It was found that the effect of T. harzianum and different leaves powder (C. fistula and A. indica) on reducing sugar of tomato plant grown under the stress of A. solani was significantly (P ≤ 0.05) decreased by 78% (A. solani) in comparison with negative control. However, the application of T. harzianum + A. solani, C. fistula + A. solani, A. indica + A. solani, T. harzianum + A. indica + A. solani, and T. harzianum + C. fistula + A. solani, respectively, significantly increased the reducing sugar of the tested plant by -38%, -97%, -74%, -188% and − 224% (Fig. 4).

Biochemical Parameters

It was investigated that the impact T. harzianum and different leaves powder (C. fistula and A. indica) on the total protein content of positive control tomato plant grown under the stress of A. solani was significantly (P ≤ 0.05) increased by -422% over negative control. However, the total protein content of tested plant was significantly decreased by 28%, 22%, 34%, 60% and 56% with the application of T. harzianum + A. solani, C. fistula + A. solani, A. indica + A. solani, T. harzianum + A. indica + A. solani and T. harzianum + C. fistula + A. solani, respectively as compared to positive control (Fig. 5).

It was found that the impact of T. harzianum and different leaves powder (C. fistula and A. indica) on the peroxidase activity of tomato plants grown under the stress of A. solani was considerably (P ≤ 0.05) increased by -722% over negative control. However, the peroxidase activity of tested plant was significantly decreased by 30%, 47%, 24%, 53% and 67% with the application of T. harzianum + A. solani, C. fistula + A. solani, A. indica + A. solani, T. harzianum + A. indica + A. solani and T. harzianum + C. fistula + A. solani, respectively as compared to positive control (Fig. 5).

It was investigated that the effect of T. harzianum and different leaf powder (C. fistula and A. indica) on the catalase activity of tomato plants grown under the stress of A. solani was significantly (P ≤ 0.05) increased by -566% over negative control. However, the catalase activity of tested plant was significantly decreased by 54%, 49%, 62%, 66% and 71% with the application of T. harzianum + A. solani, C. fistula + A. solani, A. indica + A. solani, T. harzianum + A. indica + A. solani and T. harzianum + C. fistula + A. solani, respectively as compared to positive control (Fig. 5).

Tolerance index

Furthermore, the results from tolerance index revealed (Fig. 6) that the incorporation of T. harzianum in soil either alone or in combination with the plant leaves powder i.e., C. fistula or A. indica in pathogenic infested soil exhibited 40 to 72% of tolerance in tomato plants. However, T. harzianum with A. indica and A. solani maximum tolerance index (72%) over rest of the treatments.

Principal component analysis and Pearson’s correlation analysis

The principal component analysis (PCA) exhibited the impacts of various plant extracts and T. harzianum on the growth and physiology of tomato plants grown under the stress of late blight fungi i.e., A. solani. The biplot indicated 81.28% variance (PC1 = 61.07%; PC2 = 20.21%) (Fig. 7). Likewise, PCA Pearson’s correlation also showed the positive impact on nulfying the effect A. solani on tomato plant under the soil amendment with T. harzianum and plant extracts (A. indica, C. fistula). The positive control showed negative association with growth and physiological parameters whereas exhibited positive correlation with biochemical attributes. However, soil amendment with T. harzianum and plant extract revealed positive correlation with growth attributes and physiological traits while, negative association with biochemical traits (Fig. 8).

Identification of phytochemical compounds

GCMS analysis of Cassia fistula indicated the presence of various compounds were identified. Various secondary metabolites like tannins, phlobatanins, saponins, flavonoids, terpenoids, glycosides, steroid, thymine, Linoelaidic acid and Butanoic acid, 2-oxo‐ were reported in plant. These compounds have their own medicinal importance and widely used in flock medicines to cure various disease and possess antibacterial and antifungal property (Fig. 9 and Table 2).

Table 2

Identification of phytochemicals in methanol extract of Cassia fistula using GC-MS analysis.
SrNo	RT (min)	Name of the Compound	Ref#	Mol. formula	Peak Area %
1	4.538	Benzene, chloro-	7184, 7186, 7181	C₆H₅Cl	44.55
2	6.912	Phosphoric acid tripyrrolidide	262898	C₃H₁₂N₃OP	0.30
3	6.912	Glycerin	2719	C₃H₈O₃	0.30
4	7.009	Dipropylene glycol	17309, 17306	C₆H₁₄O₃	1.14
5	7.307	1-Propanol, 2,2'‐oxybis‐	17307	C₆H₁₄O₃	0.18
6	7.932	3H-Pyrazol‐3‐one, 2,4‐dihydro‐2,	12672	C₁₀H₁₀N₂O	0.20
7	7.932	Thymine	12599	C₅H₆N₂O₂	0.20
8	8.345	Phenylethyl Alcohol	11311	C₈H₁₀O	0.26
9	8.731	4H-Pyran‐4‐one, 2,3‐dihydro‐3,5	23822	C₆H₈O₄	0.26
10	9.433	ortho tert-Butyl cyclohexyl acetate	73642	C₁₂H₂₂O₂	0.56
11	9.433	cis-2‐tert‐Butylcyclohexanol, tr.	137624	C₁₀H₂0O	0.56
12	9.433	Cyclohexanol, 2-(1,1‐dimethyleth...	137624	C₁₀H₂₀O	0.56
13	10.02	2-Hydroxybutyl acetate	73711	C₆H₁₂O	0.22
14		Butanoic acid, 3-hydroxy‐3‐methyl‐	73711	C₅H₁₀O₃	0.22
15		Pentanoic acid, 3-hydroxy‐, meth...	73711	C₆H₁₂O₃	0.22
16	12.26	Butanoic acid, 2-oxo‐	4754	C₄H₆O₃	0.44
17		1-Nonanamine	23501	C₅H₁₃N	0.44
18		2-(Isobutoxymethyl)oxirane	15445	C₇H₁₄O	0.44
19	12.305	2-O‐Methyl‐d‐xylose	39949	C₆H₁₂O₅	0.60
20	12.305	Ethanol, 1,1'-oxybis‐, diacetate	65180	C₈H₁₄O₅	0.60
21	12.305	1,3-Dioxolane‐2‐propanal, 2‐methyl‐	23872	C₇H₁₂O₃	0.60
22	13.928	9,11-Octadecadiynoic acid, 8‐hyd...	207231	C₁₉H₃₄O₂	0.59
23	13.928	Silane, ethyltrimethyl-	4950	C₅H₁₄Si	0.59
24	13.928	N,N-Dimethylacetamide	2114	C₄H₉NO	0.59
25	14.211	Thiophene, tetrahydro-2‐methyl‐	4909	C₅H₁₀S	4.26
26		Methyl 4-O‐methyl‐d‐arabinopyran...	53235	C₇H₁₄O₅	4.26
27		Thiophene, 2-ethyltetrahydro‐	9353	C₆H₁₂S	4.26
28	14.250	3-Methyldodecanoic acid	92084	C₁₃H₂₆O	0.95
29		• 3-O-Methyl-D-fructose	69600	C₇H₁₄O₆
30	14.275	• Tetrahydrothiophene	4909	C₅H₁₀S	1.00
31		4-O‐Methylmannose	69596	C₇H₁₄O₆
32		3,4-Di‐O‐methyl‐L‐arabinopyranose	53233	C₇H₁₄O₅
33	14.327	Thiophene, tetrahydro-2‐methyl‐	4909	C₅H₁₀S	1.44
34		4-O‐Methylmannose	69596	C₇H₁₄O₆
35		Thiophene, 2-ethyltetrahydro	9353	C₆H₁₂S
36	14.636	n-Hexadecanoic acid	143511	CH₃(CH₂)₁₄COOH	4.35
37		Pentadecanoic acid	126203	C₁₅H₃₀O₂
38	15.003	3-Pentanol, 2,3,4‐trimethyl‐	15777 0	C₈H₁₈O	8.14
39	15.057	2-(2-Butoxyethoxy) ethanol	243499	C₈H₁₈O₃	2.92
40		Methyl alpha-D-Glucopyranoside	53243	C₆H₁₂O₅
41		1,3-Dioxolane, 2‐(3‐methoxypropy	36720	C₈H₁₆O
42	15.916	Methyl alpha-D-xylopyranoside	53243	C₆H₁₂O₅	8.52
43		Thiophene, 2-ethyltetrahydro	9353	C₁₁H₂₂O
44		2-[2‐[2‐(2‐Butoxyethoxy)ethoxy]e	188598	C₁₀H₂₂O₄
45	15.229	alpha.-D‐Xylofuranoside, methyl	53243	C₆H₁₂O₅	2.59
46		Methyl 4-O‐methyl‐.beta.‐D‐xylop	53239	C₇H₁₂O₆
47	15.258	4-Methylmannose	69596	C₇H₁₄O₆	1.92
48		1,3-Dioxolane, 2‐(3‐methoxypropy	36720	C₈H₁₆O
49	15.288	.alpha.-D‐Xylofuranoside, methyl	53243	C₆H₁₂O₅	2.77
50		4-O‐Methylmannose	69596	C₇H₁₄O₆
51	15.326	alpha.-D‐Xylofuranoside, methyl...	53243	C₆H₁₂O₅	2.71
52	15.352	Thiophene, 2-ethyltetrahydro	9353	C₆H₁₂S	4.50
53		alpha.-D‐Xylofuranoside, methyl...	53243	C₆H₁₂O₅
54	15.923	Octadecanoic acid	179093	C₁₈H₃₆O₂	2.64
55		Linoelaidic acid	173566	C₁₈H₃₂O₂
56	18.417	Hexadecanoic acid, 2-hydroxy‐1	236864	C₁₉H₃₈O₄	0.10
57		15-Hydroxypentadecanoic acid	145878	C₁₅H₃O₃
58		Glycerol 1-palmitate	236855	C₁₉H₃₈O₄
59	19.411	Supraene	308259	C₃₀H₅₀
60		2-Dimethyl 1-3-(3, 7, 12- trimethgyl –hexadeca-3,7,11,15-tetraenyl)-oxirdane	316556	C₃OH₅₀O

Contemporary study was conducted to determine the effect of A. solni on the growth of tomato plant, moreover disease was controlled by using two plant powders and antagonistic fungi i.e., C. fistula, A. indica and T. harzianum. The morphological alternations in tomato plants were observed after 20–25 days of seedlings. Findings indicated that the growth parameters of tomato seedlings were significantly declined when infested by early blight disease. The shoot and root length of the plant gradually declined up to 55%, 47.5%. It was observed that plant showed the symptoms of stunned growth, wilting of leaves, decrease in shoot and root ratio, reduction in leaves number and over all plant biomass. The current findings are in harmony with many previous reports, which indicated that A. solni decrease the overall yield and growth of tomato plant. Previous studies demonstrated that (21) dark-colored bullseye spots with a necrotic center in a concentric ring pattern was appear in tomato plant, when lesions get larger, leaves turn blighted and started to fall. Because of this, tomato fruits eventually lose their foliage and become more susceptible to sunburn, which reduces their output and quality.

Biochemical parameters indicated that the total chlorophyll content and reducing sugar level in positive control (Plant infested with A. solni) significantly reduced up to 78% as compared to negative control (plant without fungus). According to previous (22) studied indicated that chlorophyll content is reduced when fungus attacked on plant, it might be due to the excessive production of toxin compounds by A. solani. Furthermore, he concluded that toxin compounds like TeA, AoH and AME were produced during infection that might disturb the photosynthesis content resulting in less sugar content in infected plant.

However, the improvement in growth was observed when a mixture of plant powders with Trichoderma was applied. The growth was enhanced in stem and root up to 87% (T. harzianum + A. indica) and − 75% when (T. harzianum + C. fistula) were applied. during his work with A. solni and Trichoderma (23) resolved that the mycelium growth of A. sloni fungus was declined when grown in a medium, and culture assay indicated that Fungus growth is actively inhabited by T. harzianum. While working with M. phaseolina and C. fistula flower extract (24), they concluded that flower shown marvelous positive effect against fungal infection, GCMS analysis of C. fistula flowers indicated the presence of a Total of 37 compounds among the most prominent are, Hexacosane, heptacosane, and benzene, 1, 2, 3-trimethyl- from chloroform fraction. These compounds derived from flower extract of Amaltas showed anti-microbial activities against M. phaseolina growth. Our results indicated that Cassia leave powder induced positive results up to 3to 4 folds against A. solni growth, this might be the presence of antimicrobial compounds in leaves. On the other hands both medicinal plants A. indica and C. fistula play an important role in providing protection against fungal stress in plants. GCMS analysis of Cassia indicated that various compounds were present in leaf powder of plant. Same result was found by (25) Altameme et al. (2015), during GCMS analysis he concluded that various antifungal compounds are present in leaf of Cassia fistula that help to eradicate fungal disease and protect plants from fungal attack as well.

Additionally, when the plant is treated with plant leaves powder separately and in combination with Trichoderma fungi, the results were fascinating. The total chlorophyll content was significantly increased up to -130% in tomato plant, when the combination of C. fistula and T. harzianum were applied. The same result was observed by (26), according to him it might be the excessive production of specific enzymes and hormones that produce in stress condition and help the plant to grow by suppressing the growth of pathogen fungi.

The results also revealed that the Trichoderma presence in both C. fistula and A. indica leaves powder, had enhanced the overall growth parameters, whereas when these powders were used separately, the plant suffered from stunted growth. It is suggested that this might be the presence of melatonin in Trichoderma that acts as a development agent and promotes plant growth by eradicating A. Solani growth. This hypothesis is proven (27) while working with five stains of Trichoderma, found out the presence of melatonin by using the chromatography technique. Melatonin is a hormone, resemble to IAA that promotes cell growth, cell division, and chlorophyll content and acts as a scavenger for ROS species in plants.

Present studies revealed that despite bactericides, pesticides, and fungicides, plants secondary metabolites are the alternative source of various compounds. These compounds have highly antimicrobial activities (28). Cherkupally et al, showed that using plant extracts as a medication could be more affordable, accessible, safe for the environment, and non-polluting. It is significant to look for more recent drugs that have less resistance (29). Drugs derived from natural sources are essential for the management and mitigation of human diseases. In many developing countries, traditional medicine is one of the primary kinds of healthcare. The medical benefits of herbs are well known, and they are frequently utilized in traditional medicine (30).

Various fungicides are widely available in local markets, these products are costly and cause environmental pollution. Furthermore, these chemicals enter into the food chain and result in biomagnification. Whereas, using plant leaves powder in combination with antagonistic fungi is an alternative approach due to an environment-friendly, biodegradable process. The present study indicated that Trichoderma harzianum and plant leaves i.e., Cassia Fistula and Azadirachta indica powder can be used to control early blight of tomato caused by Alternaria solani. Results indicated that bio-agents can be a ray of hope in enhancing tomato growth by eradicating the fungal pathogen growth.

Funding statement

The Authors would like to acknowledged research supporting project number RDP2024R393, King Saud university, Riyadh, Saudi Arabia.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

No, I do not have any research data outside the submitted manuscript file. Not applicable (this manuscript does not report data generation or analysis).

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable

Authors contributions

A: Ayesha Shafqat: Proof reading

B: Sundus Akthar : Methodology preparation

C: Rubina Shakir: Whole experiment performance

D: Anis Ali Sha: Editing and finalization

E: Natasha Hassan: Original draft preparation

F: Anza Afzal : GCMS analysis , data interpretation

G: Mansour K. Gatasheh : Biochemical analysis of all treatments

H: Shifa Shaffique : Statistical Analysis PC1 PC2, correlation, LSD

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Mitigation of Early Blight in Tomato Plant: Evaluating Trichoderma harzianum, Cassia fistula and Azadirachta indica leaves powder

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Abstract

Figures

Introduction

Material and methods

Isolation and Identification of Fungus

Plant material preparation

Tolerance index

Physiobiochemical assays

Total Chlorophyll Content (TCC)

Total Protein Content (TPC)

Reducing Sugar (RS)

Catalase activity (CAT)

Peroxidase Detection (POD)

GCMS Analysis

Statistical Analysis

Results

Morphological Alternations

Growth Parameters

Shoot parameter

Root parameters

Physiological Parameters

Biochemical Parameters

Tolerance index

Principal component analysis and Pearson’s correlation analysis

Identification of phytochemical compounds

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

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