Impact of Biocontrol Agents on Biochemical Changes of Aquatic Weed Water Hyacinth, Pontederia crassipes (Mart.) Solms-Laubach

doi:10.21203/rs.3.rs-4160435/v1

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Impact of Biocontrol Agents on Biochemical Changes of Aquatic Weed Water Hyacinth, Pontederia crassipes (Mart.) Solms-Laubach

https://doi.org/10.21203/rs.3.rs-4160435/v1

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Invasive weeds are rapidly adapting to evolutionary changes, brought about by exposure to the plethora of plant-antagonist’s interactions, which eventually leave an effect on the biological control of the weed. These interactions create an array of biochemical responses in the plant, which induce a range of defensive mechanisms to reduce the threat of injury. The present investigation reports the role of plant secondary metabolites in plant defense that may involve deterrence of antifeedant activity associated with the application of biocontrol agents against Pontederia crassipes. Generally, an infestation is preferred on non-challenged plants, as increased content of alkaloid, phenol, or tannin, which Pontederia crassipes produces when infested by its agents, deters the latter by providing a toxic unpleasant atmosphere. Variation in flavonoid level also brings about some physiochemical changes in the weed which impede the entry of phytopathogens. When a few metabolites are used to dissuade the agents, some, like glycosides, attract feeders to lay eggs and allow their population to flourish. This study concentrates on the signals that enable P. crassipes to recognize and respond to the attack and measure the effect in biochemical terms. Through this has, an overall outlook of the fitness costs of attack not only for the weed but over the range of trophic levels has been enlightened with more scope to understand the underlying mechanisms, before the multi-agent release of agents.

multitrophic interaction

water hyacinth

plant secondary metabolites

Fusarium oxysporum

Neochetina spp

Under the rapidly changing environmental conditions, plants are exposed to a wide range of biotic interactions, which include insect and pathogens attack. Hence plants have evolved multiple defense mechanisms by which they are able to cope or become resistant to various kinds of biotic and abiotic stress that help to retain their fitness (Ballhorn et al. 2009; Mitchell et al. 2016). Secondary metabolites (SMs) are the products formed by interactions with the environment during plant growth and development. Many plant SMs act as chemical defenses against herbivores(Fraenkel 1959) and the presence of such metabolites has facilitated coevolution between plants and herbivores (Ehrlich and Raven 1964; Berenbaum et al. 1992). Ecological analysis of the costs and benefits of chemical defenses has allowed ecologists to make predictions about the number of chemical defenses in plants. Haukioja and his co-workers proposed a model of plant defense, which suggests that these ‘induced defenses’ reflect the ability of meristematic tissues to compete for resources that in turn could be allocated to growth and defense (Haukioja 1991; Honkanen et al. 1994). Fast-growing plants (species or individuals) synthesize smaller amounts of SMs than slow-growing plants because the replacement of plant materials lost to herbivores is more difficult in slow-growing plants (Feeny and Bostock 1968; Janzen 1974; Rhoades and Cates 1976; Coley et al. 1985; Bazzaz et al. 1987; Herms and Mattson 1992).

Most studies aim at understanding one-to-one interaction, but plants are under the influence of multiple organisms that are constantly interacting with each other, affecting not just the plants’ responses but also how other co-existing species respond to each other (Ray and Hill 2016; Dutta and Ray 2017). Associations among vascular plants, fungi, and insects have a long history (Dutta et al. 2015). Aromatic compounds accumulating in the host tissue of plants affected by parasites, like fungi, bacteria or viruses, is a widespread phenomenon (Farkas and Kiraaly 1962). Fungal infection of plants usually alters plant chemistry, by either increasing defense-chemical levels or decreasing nutrient levels, which, in turn, can influence insect performance (Raman et al. 2012). Pathogenic fungi are associated with plants either as biotrophs or as necrotrophs. Necrotrophs extract nutrients by killing host plant tissues, whereas biotrophs extract nutrients without doing so. Both induce specific, altered physiologies in their host plants. Such changes may affect the growth and development of insects negatively (Tasin et al. 2012). Plant-feeding insects and plant-pathogenic fungi often co-occur on the same plants (Karban et al. 1987). When infected by fungi (e.g., Botrytis cinerea Pers., Sclerotiniaceae), Vitis vinifera L. (Vitaceae) leaves synthesize SMs, pathogenesis-related proteins, chitinase, and β-1, 3 glucanase (Trotel-Aziz et al. 2006). In contrast, fungal infection can suppress plants’ defense responses by altering secondary-metabolic pathways and improving nutritional quality, rendering the plant amenable to insect colonization (Cardoza et al. 2003). Pathogenic fungi modify plant volatiles and their profiles (Witzgall et al. 2012). Many SM-like cyanogenic glycosides and secondary glucosinolates occur as inactive precursors and become active in response to tissue damage or pathogen attack. This activation often involves enzymes, which are released due to a breakdown in cell integrity (Osbourn 1996; Dutta 2021).

Water hyacinth (WH), Pontederia crassipes Mart. Solms-Laubach (Pontederiaceae), a native of tropical South America, is, one such serious aquatic weed (Holm et al. 1977), on which multiple phytopathogenic and arthropodal biocontrol agents have been released(Coetzee et al. 2011) but scanty reports portray the scenario of the biochemical changes or the mechanisms that might be occurring as a result of the release of multiple agents (Dutta and Ray 2017). Several studies have shown synergistic effects on the release of two or more biocontrol agents (Ray and Hill 2012), but contrary antagonistic results with the release of multiple agents can make the biocontrol process less effective or even riskier (McEvoy and Coombs 2000; Dutta and Ray 2017). Therefore, to understand how phytopathogens may alter the chemistry of the host plant, making it favorable for herbivore consumption or leaving a direct impact on herbivore(Dutta and Ray 2017) biochemical analysis of affected and non-affected P. crassipes, prior to the release of agents may be useful. With the challenge of controlling the infestation of the target weed, understanding the underlying mechanisms can enlighten us to realize the perfect combinations of the biocontrol agents to be released against the target weed.

In this study, we have focused on various secondary metabolic profiles with respect to the stress undergone by WH in response to its biocontrol agents. The experiments were conducted on different sets of WH reared separately and infested with water hyacinth weevil Neochetina bruchi (Hustache) (NB)(Coleoptera: Curculionidae) and mite Orthogalumna terebrantis Wallwork (Acarina: Galumnidae) which are arthropod agents (AD). Fusarium oxysporum Schlecht (FO) was used as the fungal biocontrol agent (Dutta 2021). The SMs including phenols, glycosides, tannins, and flavonoids were studied in response to the mentioned biocontrol agents and their effect was evaluated over the period of a year.

Collection and maintenance of plant

Fresh, insect and disease-free WH in their early growth stages (4–6 leaves per plant, 15–25 cm in length, from shoot to the tip of the root) were collected from the field surveys from various waterbodies of Kolkata and its outskirts. They were transferred into a big 1000 L tank in the greenhouse as the stock culture at Presidency University, Kolkata, India. All the tanks were filled with tap water and supplemented with farmyard manure and 15–3–12 N: P: K, slow-release fertilizer. The plants were acclimatized before using them for the experiment.

Fungal pathogen and insects used for the study

Cultures of fungi Fusarium oxysporum Schlecht (FO) were isolated from diseased leaves of WH and collected during periodical surveys of various water bodies in and around Kolkata (Dutta et al. 2023). They were selected for this study because they have an excellent potential for causing disease against water hyacinths under controlled conditions (Dutta 2021). A 14-day-old culture of the fungus grown in a Biological Oxygen Demand (BOD) incubator at 27^oC and 12h photoperiod was used for all the experiments.

Neochetina bruchi (Hustache) (Coleoptera: Curculionidae) (NB), the water hyacinth weevil, and Orthogalumna terebrantis Wallwork (Acarina: Galumnidae) (OT), the water hyacinth mite, were collected from arthropod infested WH in local water bodies. The arthropods were reared separately in several 500 L tanks under greenhouse conditions. The tanks containing weevils were covered with fine mesh nets to prevent their escape. The second set of tanks, where mite was maintained, were placed at a distance, to prevent their invasion of weevil-reared tanks. The tanks were replenished with water and fertilizers (NPK) for plant maintenance as and when required. All dead leaves, stems and daughter plants were removed from time to time to maintain a healthy culture. Adult insects were collected from these stock cultures for experimentation.

Application of spore suspension and arthropods individually and in combination

The fungal conidial suspension was formulated to contain about 10⁵ − 10⁷ spores/ mL using a hemocytometer. To this Tween 20 (Oxysorbic polyoxyethylene sorbitan monooleate) was added at the rate of 0.05 mL/ 50 mL of fungal suspension as a surfactant (Ray). Freshly prepared spore suspension of the fungus was applied to WH. Arthropod-damaged (AD) WH leaves were collected from their respective stock cultures. The conidial suspension was applied on WH in various treatments, in combination with N bruchi (NB) and O. terebrantis (OT) against the weed. Experimental control was taken, which included fresh unaffected plants, with no biocontrol agents. The selected fungi (FO) and two arthropods were applied on WH plants in different combinations of FO + WH, OT + WH, NB + WH, FO + AD + WH and WH (FO – Fungus infected; OT – mite damaged; NB - weevil damaged; AD – both mite and weevil damaged; WH – undamaged and uninfected water hyacinth). The experiment was terminated after 30 days of application of biological control agents. The plants were taken out from the experimental tanks and washed gently with water and sent to the laboratory for further studies.

Preparation of plant sample

Biochemical studies were undertaken using the treated plants (with arthropod and fungal biocontrol agents) and untreated fresh plants. The root portions were cut-off, and the remaining parts were washed thoroughly under running tap water to free them from debris. The leaves and shoot portion of the weed were chopped into small pieces and dried in shade and then in a hot air oven (at 40^o C). The dried plants were made into a fine powder using a mixer grinder. The leaf powders of the test weed were stored for further studies.

Preparation of plant extracts

The powdered plant parts from each treatment were separately extracted with 95% ethanol, 95% methanol and distilled water, overnight at 4^oC. Samples for glycoside content were extracted using chloroform and chilled overnight at 4^oC. The extracts were then filtered using Whatmann No. 1 filter paper (42.5 mm Circle) and the solutions were concentrated using rotary evaporator (Buchi 100v Rotavapor), following their respective temperature and pressure conditions (Table 1). The crude samples isolated were then used for performing the tests (Lalitha et al. 2012).

To achieve optimal distillation results with a rotary evaporator, it is important to consider the specific solvent being used. The following guidelines can be followed:

Water Bath Temperature: Set the water bath temperature to 60°C. There is no need to exceed this temperature as it can potentially degrade the solvent or lead to unnecessary evaporation.
Cooling Water Temperature: Ensure that the cooling water temperature is below 20°C. This helps to efficiently condense the evaporated solvent vapor, facilitating its collection and preventing excessive loss.
Vacuum Adjustment: Adjust the vacuum level based on the boiling point of the solvent being distilled. The boiling point determines the required vacuum to achieve efficient evaporation. Refer to the list below for recommended vacuum levels corresponding to different solvent boiling points:

For solvents with a boiling point below 40°C, adjust the vacuum level accordingly to facilitate their evaporation.

For solvents with a boiling point at or around 40°C, ensure the vacuum is set at an appropriate level to maintain efficient distillation.

For solvents with a boiling point above 40°C, a lower vacuum level may be sufficient to achieve the desired distillation.

Table 1

Solvents and its vacuum boiling point maintained
	Vacuum for boiling point at 40^o C
Solvent	(mbar)
Ethanol	175
Methanol	337
Distilled Water	72

Tests for alkaloids

Dragendroff’s test: The extract on treating with a few drops of Dragendroff’s reagent (Millipore Sigma), formation of orange, brown precipitate confirms the presence of alkaloids (Adams and Camp 1966).

Tests for flavonoids

Sodium hydroxide (NaOH tests): 1 ml of the stock solution was taken in a test tube and a few drops of 4%NaOH solution was added to it. The appearance of an intense yellow color in the test tube, which goes colorless with the addition of a few drops of dilute acid, indicated the presence of flavonoids (Mahesh et al 2013.)

Test for glycosides

Concentrated H₂SO₄ test: In 5 ml of extract, 2 ml of glacial acetic acid, one drop of 5% Ferric chloride (FeCl₃) and conc. Sulphuric acid (H₂SO₄) was added. The brown ring coloration in interphase marked the presence of glycosides(Gumbinger et al. 1992).

Ferric chloride (FeCl ₃ ) test: The extract was treated with 2 ml of water and 10% aqueous, freshly prepared, FeCl₃ solution and observed for blue or green coloration (Hirakura et al. 1986).

Tests for tannins

Lead acetate test: The ethanol extract was treated with a few drops of 1% lead acetate solution (w/v) and observed for the formation of a yellow or red precipitate (Wall et al. 1969).

Ferric chloride test: Blue or black precipitate formation on treating the ethanol extract with 2 ml of freshly prepared FeCl₃ solution confirms the presence of tannins (Mailoa et al. 2013).

Tests for terpenoids

The qualitative phytochemical analysis for the presence of terpenoid was determined by using 0.8 g of plant sample, taken in a test tube, and 10 mL of 95% methanol poured into it. The mixture was shaken well and filtered. 0.5 mL extract of plant sample and 2 mL of 99% chloroform were mixed, and 3 mL of concentrated H₂SO₄ (98% v/v) was then added to it. The formation of reddish-brown color indicates the presence of terpenoids in the selected plants (Rao et al. 2021).

Quantitative analysis

Estimation of alkaloids

The methanol-filtered extract was evaporated on Buchi100v Rotavapor under vacuum at a temperature of 40^oC to dryness. A part of this residue was dissolved in 2 N Hydrochloric acid (HCl) and then filtered. 1 mL of the solution was transferred to a separatory funnel and washed with 10 mL (about 0.34 oz) chloroform (3 times). The pH of this solution was adjusted to neutral with 0.1 N NaOH. Then 5 mL of bromocresol green solution (BCG), was prepared (Tabasum et al. 2016]). 5 mL of phosphate buffers and 5 mL of freshly prepared BCG were added to the solution.

For the preparation of standard (Figure S1A), aliquots of caffeine standard solution (made by dissolving 1 mg pure atropine in 10 mL distilled water) were measured and transferred to a separatory funnel and 5 mL (about 0.17 oz) of pH 4.7 phosphate buffers and 5 mL of BCG were added.

The mixtures, in both cases, were shaken and the complex formed was extracted with 1, 2, 3- and 4 mL chloroform by vigorous shaking. The extracts were collected in a 10 mL volumetric flask and diluted to volume with chloroform. The absorbance of the complex in chloroform was measured at 470 nm against a blank prepared above without caffeine.

Estimation of flavonoids

Flavonoids were estimated based on the formation of the flavonoids-aluminium complex. 1 mL of the extract was mixed with 0.075 mL of 5% sodium nitrite solution and incubated at room temperature for 10 min (Zhishen et al. 1999). 10% Aluminum Chloride (AlCl₃) was then added and incubated at room temperature for 6 min. Later 1 N NaOH was added to it. The absorbance was read at 510 nm against a blank reagent. The absorption of standard quercetin solution (following serial dilution) was measured under the same conditions (Figure S1B)

Estimation of glycosides

10 mL of the extract and 10 mL of Baljet’s reagent (5ml of 1% of picric acid solution in ethanol and 5 ml of 10% alcoholic potassium hydroxide solution). were taken and allowed to stand for an hour, then the solution was diluted with 20 mL distilled water and mixed. An orange-red color complex appeared. The intensity (absorbance) of color produced is proportional to the concentration of glycosides which is measured against the blank at 495 nm using a spectrophotometer. The difference between the test and the control was taken for the calculation.

Concentration (%) =(Absorbance*100)/17 g %

For standardization, 0.02% digitoxin was prepared in chloroform: methanol (1:1) and the graph was plotted using various concentrations (Figure S1C.).

Estimation of total phenols

Total phenols were estimated using Folin - Ciocalteau method (Bray and Thorpe 1954). 1 mL of the extracted solution was transferred into a test tube, to which 0.5 mL of 2 N Folin - Ciocalteau reagent was added and incubated at room temperature for 3 min. Further 1.5 mL of 20% Sodium Carbonate (Na₂CO₃) was added after 3 min and the volume was made up to 8 mL with distilled water, followed by vigorous shaking and incubating in a boiling water bath for 1 min. Rapidly cooled and the absorbance was read at 650 nm against blank reagent. The data were used to estimate total phenols using a standard calibration curve obtained from various diluted concentrations of gallic acid, measured at 765 nm (Figure S1D), after which the results were expressed as mg/g sample.

Estimation of tannins

500 mg of the sample was weighed into a 200 mL plastic bottle. 50 mL of distilled water was added and mixed thoroughly for 1 h in a shaker. This was filtered into a 50 mL volumetric flask and made up to the mark. Then 5 mL of the filter was pipetted out into a test tube and mixed with 2 mL of 0.1 M FeCl₃ in 0.1 N HCl and 0.008 M potassium ferrocyanide (C₆FeK₄N₆). The absorbance was read at 120 nm within 10 mins(Van Buren and Robinson 1969) The standard calibration was obtained using different concentrations of gallic acid, measured at 765 nm, following which the optical density (OD) values measured, were expressed in mg/mL (Van Buren and Robinson 1969). (Figure S1D).

Estimation of terpenoids

The previously prepared sample for qualitative analysis was transferred from the assay tube to a colorimetric cuvette [95% (v/v)], methanol to be used as blank to read absorbance at 538 nm. For the standard curve, 200 µl of previously prepared linalool solution in methanol was added to 1.5 mL of chloroform and serial dilution was done, where volume make-up was done by the addition of 95% (v/v) methanol (Figure S1E).

Statistical Analysis

Each set of treatments was replicated six times. Statistical analysis was conducted with R version 4.2.2 in platform aarch64-apple-darwin20 (64-bit). Shapiro-Wilk test and histogram confirmed (P < 0.05) rejection of the data normality null hypothesis among all samples. Kruskal-Wallis Rank Sum test non-parametric method to evaluate overall significant differences (P < 0.05) among treatments (Control, Field, Fungus, Fungus + Insect and Insect). Dunn’s test (1964) pairwise multiple comparisons with no P-value correction as a post hoc method to identify significant differences (P < 0.05, P < 0.01, P < 0.001) between treatments. Correlation between treatment solvents was quantified through Spearman’s rank correlation, only strong positive and/or negative relationships (-0.7 > R > 0.7) were used for supplementary visualizations.

Qualitative analysis

Phytochemical screening of methanol extracts of P. crassipes showed the presence of all tested active constituents, whereas ethanol and aqueous extract selectively detected their presence (Table 2). Ethanol extract showed the presence of all tested metabolites (alkaloids, tannins, phenol, glycosides, flavonoids and terpenoids), except tannin, whereas aqueous extract could not confirm the presence of alkaloids and flavonoids. The presence of glycosides was detected in the chloroform extract of the study plant samples.

Table 2

Qualitative analysis of metabolites in ethanol, methanol, aqueous and chloroform extract of P. crassipes. (+) shows positive yield in the solvent (-) shows negative yield in the solvent. (NA) -Not applicable shows the solvent not used for the extraction.
Compound	Ethanol (E)	Methanol (M)	Aqueous (A)	Chloroform (C)
	(Solv)	(Solv)	(Solv)	(Solv)
Phenol	+	+	+	NA
Alkaloid	+	+	-	NA
Flavonoid	+	+	-	+
Terpenoid	+	+	+	NA
Glycosides	+	+	+	+
Tannin	-	+	+	NA

Quantitative analysis

Table 3 displays the optimal quantitative yields of metabolites using four distinct solvents: ethanol (E), methanol (M), chloroform (C), and aqueous (A). Notably, ethanol exhibited the highest yields for phenol alkaloids, flavonoids, and terpenoids. For tannin, quantitative analysis was exclusively conducted using aqueous solvent. Remarkably, glycoside was the only metabolite that yielded measurable results in all four solvents utilized in the study.The quantitative analysis showed variable results (Fig. 1) among the SMs identified from water hyacinth in various solvents like methanol (M), ethanol (E), chloroform (C) and aqueous (A). The phenolic content in weevil-fed water hyacinth (NB + WH) was evidently higher than in comparison to fungus (FO + WH) infected (M = 54.248 mg/mL; E = 57.408 mg/mL; A = 56.16 mg/mL) and FO + AD + WH (fungi-arthropod infested water hyacinth) (M = 40.387 mg/mL E = 37.195 mg/mL A = 43.975 mg/mL), as observed from both the alcoholic (M = 77.401 mg/mL, E = 78.213 mg/mL) and aqueous extraction (75.717 mg/mL) (Fig. 1a).

Ethanol extract of water hyacinth showed the highest alkaloid activity, over methanol and aqueous extracts. From the ethanolic (E) and aqueous (A) extract, the alkaloid content was observed to be maximum, with 0.214 mg/mL and 0.175 mg/mL respectively, when infested with FO + AD + WH, than in undamaged (WH) or FO + WH (E = 0.193 mg/mL; A = 0.124 mg/mL) or NB + WH (E = 0.159 mg/mL; A = 0.162 mg/mL) treated leaves separately. But the methanol (M) extract showed a decline in the alkaloid content with an increase in damage to the weed with both FO + WH (0.104 mg/mL) and FO + AD + WH (0.109 mg/mL) showing a higher drop than just NB + WH (0.128 mg/mL) damaged leaves (Fig. 1b).

Damaged leaves showed higher flavonoid content than control leaves (WH = 0.199 µg/mL) of WH. The methanol extract showed NB + WH leaves (0.209 µg/mL) have more flavonoid content than FO + WH (0.201 µg/mL), and the ethanol extract showed the same trend as methanol (FO + WH = 0.203 µg/mL, FO + AD + WH = 0.202 µg/mL) (Fig. 1c).

The alcohols proved better (0.048mg/mL), in extracting terpenoids from water hyacinth as compared to aqueous extract (0.047 mg/mL) (Fig. 1d).

The glycosidic content increased in NB + WH leaves (M = 0.586 mg/mL; E = 0.565 mg/mL) than in WH (fresh unaffected leaves) (M = 0.493 mg/mL; E = 0.481 mg/mL), followed by FO + WH (M = 0.489 mg/mL; E = 0.474 mg/mL) and FO + AD + WH (M = 0.390 mg/mL; E = 0.325 mg/mL) treated water hyacinth (Fig. 1e).

Tannin did not show a noticeable change when extracted in methanol, though its presence was detected in the qualitative test. The quantitative value was estimated in water (A) for tannin. The level of tannin content increased in damaged leaves of WH, maximising in FO + AD + WH treatments with 1.931 mg/mL of tannin, and FO + WH (1.572 mg/mL) respectively (Fig. 1f).

The best yield of the different metabolites in the different sets of treatments (WH, OT + WH, FO + WH, NB + WH and FO + AD + WH) are highlighted using boxplots (Fig. 2) showing the statistically significant differences among the different solvents methanol, ethanol, aqueous, chloroform for the SMs’ extraction. No significant differences between the solvents and the treatment groups were observed (Fig. 2b and Fig. 2d). There are significant differences noticed in phenol, flavonoid, glycoside and tannin. The density histogram (or densigram) shows the highest concentration of each solvent (Fig. 2a, Fig. 2c, Fig. 2e and Fig. 2f).

The correlation analysis showed that the extraction of methanol and ethanol in phenol and glycoside are closely related and have a similarity of 0.87 and 0.85. Also in glycoside, methanol and ethanol have 0.74 and 0.80 co-efficient with aqueous similarity respectively indicating the yield to be similar (Figure S2A, S2B, S2C, S2D). The correlation analysis gives us a vivid idea about the yield of metabolites irrespective of the different kinds of solvents used for extraction in different groups of treatments.

Table 3

Quantitative yield of metabolites in ethanol, methanol, aqueous and chloroform extract from *P. crassipes*
	Ethanol	Methanol	Aqueous	Chloroform
Compound	(Solv)	(Solv)	(Solv)	(Solv)
Phenol	+++	+++	++	-
Alkaloid	+++	+	+	-
Flavonoid	+++	+	+	-
Terpenoid	+++	+++	+	-
Glycosides	+	+	+	+
Tannin	-	-	+++	-

Solv: solvent, +++: Highest amount of yield, ++: Moderate amount of yield, +: Lowest amount of yield, -: Lack of yield.

Plants face various biotic and abiotic challenges such as diseases, herbivores, and water and nutrient deficiencies that impact their overall growth significantly (Misra et al. 2023). In response to such biotic and abiotic stresses, multilayered defense strategies have been incorporated during this magnanimous evolutionary time scale (Ahmad et al. 2022). Classical weed biocontrol also depends a lot on ecological interactions and the evolutionary history of specialist herbivores and their host plants to manage invasive plants in their introduced range (Dutta et al. 2023). The secondary biochemistry of plants plays a significant role in the evolution of plant-arthropods-fungi relationships, along with the shared history of co-evolution and complementation which leads to co-existence and host-specificity (Dethier 1954).

Successful determination of biologically active compounds from plant material is largely dependent on the type of solvent used in the extraction procedure (Ellof 1998). Plant SMs are compounds that have no fundamental role in the maintenance of life processes in plants (Hartmann 1991), but they are important for the plant to interact with its environment for adaptation and defense (Misra et al. 2023). These defenses can be either ‘constitutive’, with high levels of the metabolite naturally maintained within the plant, or ‘induced’, where the metabolite is changed in abundance following herbivore or pathogen attack (Bezemer and Van Dam 2005). In higher plants, a wide variety of SMs are synthesized from primary metabolites (e.g., carbohydrates, lipids and amino acids). A variety of factors (toxins, cutinases, chitinases, cellulases, and phytoalexin demethylases) have been implicated as causal components of fungal plant disease; however, the mode of action of a fungus in pathogenicity is largely unknown (Ciuffetti et al. 1983; Kolattukudy 1985; Johal and Briggs 1992). In most cases, the interaction between fungi and plants is very intimate and the association occurs over a longer period. Under varying circumstances, distinct structures and barriers develop in the host as a response to infection, which was evident in the role of phenols in resistance to various fungi or arthropods (Harborne 1988). Fungi utilize the carbon content in the plants and convert it into their own biomass for their growth and proliferation (Huaer and Lamberti 2017).Thus, plants under low light or nutrient stress (low carbon supply) contain three folds lower concentrations of phenol than in any control or nitrogen-fed plants (Larsson et al 1986.), which hints at the drop in the phenol content level for FO + AD + WH and FO + WH treatments. Plants show proanthocyanins and even small amounts of dihydroquercetin production, involved in the defense against Fusarium species, very likely for the mentioned treatments (Skadhauge et al. 1997). Our findings thus reveal that under stress produced by any fungal infection, plants rapidly accumulate phenols at the site of infection (Matern et al. 1988), as the initial step of the defense mechanism, which penetrates microorganisms to cause considerable damage to cell metabolisms. The phenolic content for OT + WH (M = 76.991 mg/mL; E = 77.202 mg/mL) and NB + WH (M = 77.401 mg/mL; E = 78.213 mg/mL) treatments did not show a significant change as compared to undamaged treatment (WH) (Fig. 2a), though its rise in aqueous extract for NB + WH (75.717 mg/mL) was observed (Fig. 1a). This phenol resistance of water hyacinths may be one of the likely reasons for unchanged phenolic content in arthropod-treated water hyacinths, since idioblasts situated in the palisade layer contain phenolic acids which are implicated in the control of infection or attack (Galbraith 1987). Alcoholic extractions showed overall better efficacy in metabolite detection (Table 1 or 2). Ethanolic extracts for FO + WH (0.193 mg/mL) and FO + AD + WH (0.214 mg/mL) treatments showed an elevated alkaloid content, portraying an increase in endophytic colonization on the plants, which acts as an antifeedant and is toxic for other herbivores (Johnson et al. 1985). The surge in the alkaloid content of the ethanol and aqueous extract FO + AD + WH treatments, with respect to untreated and undamaged (Fig. 1b), proves how the slow introduction of multiple agents and herbivore resistance, helps the coevolution of the host plant and specialist or non-specialist herbivores (Harborne 1988).

The defense-related flavonoids can be divided into two groups: “preformed” and induced” compounds (Treutter 2005). Flavonoids induced after injury by pathogens or arthropods are a well-known phenomenon (Barry et al. 2002) tallying with the consequences that methanol extracts of affected water hyacinths. Beckman pointed out that modulating the effect on the action of Indole Acetic Acid by flavonoids might lead to changes in tissue differentiation and promotion of the formation of callus and tylose, leading to closing vessels and locking out aggressive endophytes like FO (Beckman 2000). Contrarily, ethanol extract portraying lower flavonoid content for NB + WH treatments than FO-infested ones might be a result of the sensitivity of several insects to the preformed flavonoid content or deterred by it (Widstrom et al. 2001; Haribal and Feeny 2003, Chen et al 2004) (Fig. 1c).

Terpenes are related plant SMs reported to be important factors in resistance to several insect pests and pathogens. The insecticidal activity of the terpenes is either due to their action as antifeedants (or deterrents), toxins, or modifiers of insect development (sterols such as the phyto ecdysones). Study showed terpenoid, limonene, deters Atta cephalotes L. (a leaf-cutting ant) from citrus plants (Cherrett 1972). Chemical analysis showed that terpenoid emission was inhibited more strongly in infested lima bean plants than in Brussels sprout plants after fosmidomycin treatments, showing the variation of the same terpenoid effect on different sets of plants (Mumm et al. 2008). Though the change in terpenoid content has been insignificant, alcohol proved better in extracting terpenoids (Fig. 2d). Phytoalexins (sometimes in the form of diterpenes and sesquiterpenes) are low-molecular-weight compounds that are produced as part of the plant defense system, which makes it difficult to be traced.

High glycosides concentration in AD + WH treatments was observed with higher extraction values for the alcohols (Fig. 1e, 2e). It is expected as high concentrations of plant toxins deter generalist feeders while attracting specialist biocontrol agents that either use them as a cue to locate or to accept the host plant for laying eggs and/or feeding or use them for their defense (Van der Meijden 1996; Renwick 2002). Iridoid glycosides are toxic to many generalist herbivores. Although, Nieminen with colleagues that individual plants of Plantago lanceolate L. with high iridoid glycoside concentrations in the field, favored oviposition by the specialist fritillary butterfly Melitaea cinxia L. significantly more than those plants with low concentrations (Nieminen et al 2003). Plants are cyanogenic and can form hydrocyanic acid (HCN) in response to tissue damage (D’Mello et al. 1991), associated with defense against pathogens and herbivores (Davis 1991), breaking down the cyanogenic glycosides. Healthy control plants have no detectable HCN suggesting that the cyanogenic glycosides, normally separated from the enzymes, catalyze HCN release (Osbourn 1996). Though little correlation is drawn between glycoside level and resistance to fungal pathogens reports show that high glycosidic plants are more susceptible to endophytic attack (Osbourn 1996). However, the breakdown of HCN to formamide by cyanide hydratase enzyme (Wang et al. 1992.; Fry et al. 1977) may be one reason for the low glycosidic content in FO + WH treatments, in comparison to arthropod-treated (NB + WH and OT + WH treatments) and untreated plants.

Another responsible metabolite, tannins, can defend leaves against insect herbivores by deterrence and/or toxicity. Tannins are general toxins that significantly reduce the growth and survivorship of many herbivores, reflected by the increase in the content of the metabolite in weevil) or fungi attacked water hyacinth or in their combination, thus repelling the arthropods (Fig. 1f). The defensive properties of tannins are attributed to their ability to their protein binding properties (Mazid et al. 2011).

Among all the metabolites studied, phenolics have received special attention in the study of plant-herbivore interaction, due to their presence and differentiation in large concentrations, in comparison to others. They are widely considered deterrents, antifeedants and toxins that can change the nutritional quality of plant tissues for herbivores (Lambers 1993). Although field studies suggest that the defense mechanism is overemphasized (Coley 1983), our data revealed that a. significant variation in alkaloids, glycosides, tannins, and phenols resulted in control of the weed via the biocontrol agents (arthropods and pathogens) (Fig. 1). These notable changes in metabolites and combined effect of both the pathogen and arthropods have successfully helped to control the targeted weed and instigated us for further analysis of bimolecular interaction for future designing of integrated biological control.

About 100,000 known SMs are involved in plant chemical defense systems. These phytochemicals have been shaped through the millions of years during which plants have co-evolved with their antagonists and mutualists (Wink 2008). With thousands of metabolites interplaying, their pathways and environmental interactions are difficult to understand. Although higher concentrations of SMs might result in a more resistant plant, the production of SMs is considered costly and reduces plant growth and reproduction (Thaler and Karban 1997; Siemens et al. 2002). The cost of defense has been invoked to explain why plants have evolved induced defense, where concentrations generally increase only in stressful situations (Tollrian and Harvell 1999).

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Funding:

The authors express their heartfelt gratitude to the research grant provided by the Science and Engineering Research Board, New Delhi (SERB Project No. SERB/F/5316/2013-14 dated 18.11.2013) and Department of Science and Technology (DST/INSPIRE Fellowship/2015/IF150038).

Availability of data and material

Relevant data applicable to this research are within the paper. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing Interests:

The authors declare no conflict of interest.

Author Contributions:

conceptualization, D.M., W.D., and P.R.; writing – original draft, D.M., W.D., P.R.; writing – reviewing and editing, methodology, D.M.; W.D.; D.M.; data curation and data analysis, J.C.G.P.; All authors have read and agreed to the published version of the manuscript.

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Impact of Biocontrol Agents on Biochemical Changes of Aquatic Weed Water Hyacinth, Pontederia crassipes (Mart.) Solms-Laubach

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Abstract

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Introduction

Materials and Methods

Collection and maintenance of plant

Fungal pathogen and insects used for the study

Application of spore suspension and arthropods individually and in combination

Preparation of plant sample

Preparation of plant extracts

Tests for alkaloids

Tests for flavonoids

Test for glycosides

Tests for tannins

Tests for terpenoids

Quantitative analysis

Estimation of alkaloids

Estimation of flavonoids

Estimation of glycosides

Estimation of total phenols

Estimation of tannins

Estimation of terpenoids

Statistical Analysis

Results

Qualitative analysis

Quantitative analysis

Discussion

Conclusions

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References

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