Impact of bioagents on biochemical and elemental composition of mulberry foliage infested with root-knot nematode

Introduction

Mulberry is a fast-growing perennial plant belonging to the genus Morus of the family Moraceae. It exhibits several desirable traits, including high foliage yield, a relatively short gestation period and robust environmental adaptability. Foliage is the major economic part of mulberry as the silkworm, Bombyx mori L. feeds on mulberry leaf alone to derive its nutrients for growth and productivity. Hence, the quantity of quality leaf produced per unit area has a direct bearing on cocoon production and raw silk quality. The quality of mulberry leaf is the predominant factor influencing cocoon crop success, The employment of synthetic pesticides for the management of RKNs has been associated with deleterious environmental consequences, including the persistence of toxic residues, mobility, volatility and water solubility, thereby posing a significant threat to non-target organisms (Gapasin et al., 2011) [7] . Conversely, bio-pesticides offer a sustainable alternative for plant pest and disease management, leveraging their inherent bioavailability, specificity and eco-friendly profile (Helen et al., 2016) [16] . The utilization of bioagents, comprising parasites, predators, fungi, bacteria, viruses and plant products, provides a targeted approach, ensuring the safety of non-target species, beneficial insects, higher animals, and humans, while minimizing environmental impact. Specifically, fungal and bacterial bioagents, including Purpureocillium lilacinum, Trichoderma harzianum, Trichoderma viride, Pochonia chlamydosporia and Pseudomonas fluorescens have demonstrated considerable potential in nematode management, significantly influencing growth and yield parameters (Bharath, 2022) [3] . This investigation aimed to elucidate the effects of these bioagents on the biochemical and elemental composition of mulberry foliage.

Materials and Methods

The experiment was carried out in the RKN infested mulberry garden in Kalyapura Village, Shidlaghatta Taluk, Chikkaballapura District, Eastern Dry Zone (Zone-5) of Karnataka, India at 13°14'20"N latitude and 77°52'17"E longitude, at an altitude of 904m above mean sea level. The mulberry plantation selected for the study had red sandy loam type of soil with six years old V1 variety plants, planted at the spacing of 90×90cm; bottom pruning (Kolar method) was followed; the field was irrigated in two-day intervals with drip irrigation facility i.e., based on soil moisture conditions; organic manures and inorganic fertilizers were applied following the recommended package of practice. Except for the management of RKN, the selected field was well maintained.

The following bioagents were selected and used for the management of RKN in mulberry 1. Purpureocillium lilacinum 2. Trichoderma harzianum 3. Trichoderma viride 4. Pochonia clamydosporia 5. Pseudomonas fluorescens Alongside, Nemahari (bio-nematicide developed by CSRTI, Mysore), neem cake, carbofuran 3G and control (untreated check) were included in the treatments for comparison of the efficacy.

Treatment details

Treatments

Recommendation Reference T1

Purpureocillium lilacinum 5 kg/ha (with 5 tons FYM) Saxena et al., 2021 [18] T2

Trichoderma harzianum T3

Pseudomonas fluorescens T6 Nemahari 40 kg/ha (with 400 kg FYM) Nishita and Prateeshkumar, 2015 [16]

T7

Neem cake 2000 kg/ha Dandin and Giridhar, 2014 [5]

T8

Carbofuran 3G (standard check) 40 kg/ha Dandin and Giridhar, 2014 [5]

T9

Control (untreated check) --

The bioagents were obtained from IIHR, Bengaluru. The bioagents were incorporated into the plant rhizosphere soil as per the treatment details in four replications each near the root zone of the mulberry within a week after pruning by calculating the dosage per plant.

Observations

The following parameters were recorded once before the imposition of the treatments (initial) and during the first (60 DAT) and second (120 DAT) shoot harvests in mulberry after imposing the treatments.

A composite sample of forty leaves was collected from each replication, air-dried and then dried in the hot-air oven at 60 °C for 18 hours. The samples were then powdered and stored in polythene bags. These samples were analyzed for total carbohydrates, proteins, crude fibre, ash, nitrogen, phosphorus, potassium, calcium, magnesium and sulphur contents.

The moisture content of mulberry leaves in each replication was determined by the hot air oven method, where the fresh weight of the samples was recorded and then the samples were placed in the hot air oven for 24 hours at 105±1°C, again the dry sample weight was recorded. The percent moisture content was determined by the following formula:

Leaf moisture content (%) = (Fresh weight of sample) -(Dry weight of sample)

Fresh weight of the sample × 100

To determine the leaf moisture retention capacity after six hours of harvest, a composite sample of thirty leaves was collected for each replication and the fresh weight was taken. The leaves were kept open under laboratory conditions and the weight was recorded after 6 hours of harvest. The percent moisture retention capacity was calculated by using the formula (Shivashankar, 2015) [19] :

Moisture retention capacity (%) = 100 -Moisture loss (%)

where, fresh weight is considered to be 100 percent

Where, A -fresh weight of leaf; B -leaf weight after six hours of harvest iii. Chlorophyll content (mg/g) Chlorophyll content in mulberry leaf was determined by the following procedure described. The leaf's total chlorophyll contents were computed using the formula suggested by Arnon (1949) [1] .

Total chlorophyll (mg/g) = (20.2 A645 -8.02 A663) × X 1000 × n

Where, A663 -Absorbance at a wavelength of 663 nm; A645 -Absorbance at a wavelength of 645 nm; X -Total volume of the filtrate; n -Sample weight

The total carbohydrates in the leaf samples were estimated using the Anthrone method (Hedge and Hofreiter, 1962) [9] . The amount of carbohydrates in the sample was determined by plotting a standard curve of absorbance at a wavelength of 630 nm (A630) on the Y-axis and concentration of glucose on the X-axis. The total carbohydrates is expressed in percentage.

The total proteins in the leaf samples were estimated using Lowry's method. The amount of protein in the sample was determined by plotting a standard absorbance curve at a wavelength of 660 nm (A660) along the Y-axis and protein concentration along the X-axis. The total protein is expressed in percentage

The percent crude protein was estimated using the nitrogen content determined by the micro Kjeldahl method involving digestion, distillation and titration of the sample using the formula:

Crude protein (%) = Nitrogen (%) × 6.25

The crude fibre of the sample was estimated by using moisture and fat-free sample by Fibra plus apparatus. 2 g of moisture and fat-free sample was transferred to fibre estimating thimbers and placed in the digestion chamber. 200 ml of sulphuric acid was added and the temperature was set at 500 °C for 10 min, the temperature then was reduced to 400 °C and left for 30 min., after that the acid was drained by using a suction pump and washed with boiled distilled water. After an acid wash, the digestion chamber was filled with 150 ml of boiling sodium hydroxide solution for about 30 min at 400 °C. Then the alkali solution was drained out by using the suction pump and again the tubes were washed with boiled distilled water. After that, the crucibles were placed in an oven at 100 °C for about one hour and the weight of the crucible was taken after which the crucible was placed in a muffle furnace at 550 °C for 3 hours or until the white ash was formed and the final weight was taken.

The fibre content of the sample was calculated by: Crude fibre (%) = [100 -(Moisture + Fat content of the sample)] × We -Wa Weight of the sample × 100

Where, We -Pre-weighed ash (g); Wa -Weight of dish after washing (g)

The ash content in the samples was determined using the formula (Windham et al., 1991) [22] :

Elemental constituents

The powdered leaf samples (1 g) were treated with 10 ml of concentrated HNO3 and kept overnight for pre-digestion.

Then the samples were digested with 10 ml of the di-acid mixture (9:4 ratio of HNO3 and HClO4) until the snow-white residue remained. The residue was cooled and diluted to 100 ml using distilled water, filtered and used to analyse all the elements except nitrogen.

The nitrogen percentage in the leaf samples was determined by the Kjeldahl digestion-distillation method. The plant sample (1g) was digested using the digestion mixture and sulphuric acid in a digestion flask. After complete digestion, the digested material was distilled in an alkaline medium and the liberated ammonia was trapped in a 2 percent boric acid solution containing mixed indicator. The trapped ammonia was titrated against standard sulphuric acid (Piper, 1966) [17] .

Nitrogen (%) = TV × Normality of acid × 0.014 × Volume of digested sample Weight of the sample × aliquot taken × 100

where, TV -Titration value

Phosphorus content in the di-acid digested extract was estimated by the vanadomolybdo-phosphoric yellow colour method in nitric acid medium and the colour intensity was measured at 420 nm wavelength as described by Piper (1966) [17] .

Potassium in the plant sample was determined by atomizing the diluted di-acid extract in a flame photometer as outlined by Piper (1966) [17] .

Secondary nutrients

The calcium and magnesium contents in the mulberry leaves were estimated by the EDTA titration or the versanate-titration method. The sulphur content in the di-acid digested sample was estimated by the turbidimetric method as outlined by Jackson (1973) [11] .

Estimation of micronutrients

The micronutrients were estimated using the specified reagents in the atomic absorption spectrophotometer (AAS).

Statistical analysis

The data collected from the experimental field were analyzed statistically by using one-way RCBD for testing of significance by Fisher's method of analysis of variance (Snedecor and Cochran, 1979) [20] . The level of significance used in the F-test was P = 0.05. The critical difference (CD) values were computed to compare the significance of the treatments.

Results

Leaf moisture content Among the treatments, the mulberry leaf moisture content was found to be consistent before the imposition of treatments (initial), in the intermediary (60 DAT) and also during the final observations on 120 DAT (Table 1).

Moisture retention capacity

The moisture retention capacity was found to be unwavering among the treatments before the imposition of treatments (initial). The intermediary (60 DAT) observations reveal that among the bioagents, the maximum moisture retention capacity was recorded in T. viride (97.83%) which was on par with Nemahari (97.92%) followed by T. harzianum (97.44%), P. lilacinum (97.13%), P. fluoroscens (97.05%), P. clamydosporia (96.81%), neem cake (96.40%) and carbofuran 3G (96.39%). The minimum moisture retention capacity was recorded in the untreated check (90.81%).

The maximum moisture retention capacity among the treatments on 120 DAT was recorded in Nemahari (98.5%) on par with T. viride (98.32%) followed by P. lilacinum (97.98%), T. harzianum (97.41%), P. fluoroscens (97.26%), P. clamydosporia (97.24%), neem cake (97.13%) and carbofuran 3G (96.93%). The minimum moisture retention capacity was recorded in the control (90.08%) (Table 1).

Total chlorophyll content

The total chlorophyll content was almost consistent among the treatments before the imposition of treatments (initial). Among the bioagents, the intermediary (60 DAT) observations reveal that the highest total chlorophyll content was recorded in T. viride (0.116 mg/g) on par with Nemahari (0.123 mg/g) followed by T. harzianum (0.114 mg/g), P. fluoroscens (0.112 mg/g), P. lilacinum (0.104 mg/g), P. clamydosporia (0.102 mg/g), neem cake (0.101 mg/g) and carbofuran 3G (0.099 mg/g). The lowest total chlorophyll content was recorded in the untreated check (0.062 mg/g). The highest total chlorophyll content among the treatments on 120 DAT was recorded in Nemahari (0.176 mg/g) on par with T. viride (0.170 mg/g) followed by T. harzianum (0.162 mg/g), P. fluoroscens (0.159 mg/g), P. lilacinum (0.158 mg/g), P. clamydosporia (0.155 mg/g), neem cake (0.137 mg/g) and carbofuran 3G (0.132 mg/g). The lowest total chlorophyll content was recorded in the control (0.062 mg/g) (Table 1).

Total carbohydrates

Among the treatments, the total carbohydrates in mulberry leaf was almost invariable before the imposition of treatments (initial 1).

Total protein

The total protein content was found to be unwavering among the treatments before the imposition of treatments (initial). 2).

Crude protein

The crude protein content was found to be almost consistent among the treatments before the imposition of treatments (initial). The intermediary (60 DAT) observations reveal that the highest crude protein content was recorded in T. viride (8.46%) on par with Nemahari (7.93%) followed by P. lilacinum (7.39%), T. harzianum (7.28%), P. fluoroscens (6.84%), neem cake (6.54%), P. clamydosporia (6.45%) and carbofuran 3G (5.64%). The lowest crude protein content was recorded in the untreated check (4.06%). The highest crude protein content among the treatments on 120 DAT was recorded in T. viride (13.03%) on par with Nemahari (12.31%) followed by P. lilacinum (11.92%), T. harzianum (11.65%), P. fluoroscens (10.93%), neem cake (10.43%), P. clamydosporia (9.14%) and carbofuran 3G (6.03%). The lowest crude protein content was recorded in the control (3.84%) (Table 2).

Crude fibre

The crude fibre content was found to be invariable among the treatments before the imposition of treatments (initial).

The intermediary (60 DAT) observations reveal that the highest crude fibre content was recorded in Nemahari (6.34%) on par with T. viride (6.07%) followed by P. lilacinum (5.89%), T. harzianum (5.71%), P. fluoroscens (5.57%), neem cake (5.29%), P. clamydosporia (5.18%) and carbofuran 3G (5.10%). The lowest crude protein content was recorded in the untreated check (4.22%).

The highest crude fibre content among the bioagents on 120 DAT was recorded in T. viride (8.23%) was on par with Nemahari (8.47%) followed by P. lilacinum (7.27%), T. harzianum (7.24%), P. fluoroscens (7.15%), neem cake (6.96%) and P. clamydosporia (6.82%). The lowest crude fibre content was recorded in carbofuran 3G (5.85%) followed by control (4.09%) (Table 2).

Ash

The ash content was found to be invariable among the treatments before the imposition of treatments (initial). The intermediary (60 DAT) observations reveal that the maximum ash content was recorded in Nemahari (8.00%) on par with T. viride (7.75%) followed by T. harzianum (6.75%), P. lilacinum (6.50%), P. fluoroscens (6.25%), neem cake (5.50%), P. clamydosporia (5.25%) and carbofuran 3G (4.50%). The minimum ash content was recorded in the untreated check (3.00%).

The maximum ash content among the bioagents on 120 DAT was recorded in T. viride (9.50%) was on par with Nemahari (10.25%) followed by P. lilacinum (9.25%), T. harzianum (9.00%) and P. fluoroscens (8.50%). The treatment having neem cake and P. recorded the same values for ash content (8.00%) followed by carbofuran 3G (5.00%). The minimum ash content was recorded in the control (2.75%) (Table 2).

Nitrogen

The mulberry leaf nitrogen content was found to be unwavering among the treatments before the imposition of treatments (initial). The intermediary (60 DAT) observations reveal that the maximum nitrogen content was recorded in T. viride (1.35%) on par with Nemahari (1.27%) followed by P. lilacinum (1.18%), T. harzianum (1.16%), P. fluoroscens (1.09%), neem cake (1.05%), P. clamydosporia (1.02%) and carbofuran 3G (0.90%). The minimum nitrogen content was recorded in the untreated check (0.65%).

The maximum nitrogen content among the treatments on 120 DAT was recorded in T. viride (2.08%) followed by Nemahari (1.97%), P. lilacinum (1.91%), T. harzianum (1.86%), P. fluoroscens (1.75%), neem cake (1.67%), P. clamydosporia (1.46%) and carbofuran 3G (0.96%). The minimum nitrogen content was recorded in the control (0.62%) (Table 3).

Phosphorous

The mulberry leaf phosphorous content was found to be invariable among the treatments before the imposition of treatments (initial 3).

Potassium

The mulberry leaf potassium content was found to be consistent among the treatments before the imposition of treatments (initial). The intermediary (60 DAT) observations reveal that the maximum potassium content was recorded in T. viride (0.231%) on par with Nemahari (0.233%), P. lilacinum (0.194%), T. harzianum (0.190%), P. fluoroscens (0.181%), P. clamydosporia (0.172%), neem cake (0.170%) and carbofuran 3G (0.161%). The minimum potassium content was recorded in the untreated check (0.153%). The maximum potassium content among the treatments on 120 DAT was recorded in T. viride (0.302%) on par with Nemahari (0.323%) followed by P. lilacinum (0.266%), T. harzianum (0.262%), P. fluoroscens (0.259%), P. clamydosporia (0.253%), neem cake (0.220%) and carbofuran 3G (0.189%). The minimum potassium content was recorded in the control (0.147%) (Table 3).

Calcium

The calcium content in mulberry leaves was found to be unwavering in all the treatments before the imposition of treatments (initial 4).

Magnesium

The magnesium content in mulberry leaves was invariable in all the treatments before the imposition of treatments (initial). The intermediary (60 DAT) observations reveal that the maximum magnesium content was recorded in Nemahari (0.262%) on par with T. viride (0.217%) followed by T. harzianum (0.177%), P. lilacinum (0.155%), P. clamydosporia (0.125%). The treatments having P. fluoroscens and neem cake recorded the same values for the magnesium content (0.122%) followed by carbofuran 3G (0.117%). The minimum magnesium content was recorded in the untreated check (0.107%).

The maximum magnesium content among the treatments on 120 DAT was recorded in Nemahari (0.282%) on par with T. viride (0.225%) followed by T. harzianum (0.192%), P. lilacinum (0.180%), P. fluoroscens (0.175%), P. clamydosporia (0.145%), neem cake (0.137%) and carbofuran 3G (0.127%). The minimum magnesium content was recorded in the control (0.085%) (Table 4).

Sulphur

The sulphur content in mulberry leaves was consistent in all the treatments before the imposition of treatments (initial). The intermediary (60 DAT) observations revealed that the maximum sulphur content was recorded in Nemahari (0.134%) on par with T. viride (0.118%) followed by P. lilacinum (0.109%), T. harzianum (0.107%), P. fluoroscens (0.100%), P. clamydosporia (0.096%), neem cake (0.091%) and carbofuran 3G (0.088%). The minimum sulphur content was recorded in the untreated check (0.069%).

The maximum sulphur content among the treatments on 120 DAT was recorded in Nemahari (0.185%) on par with T. viride (0.179%) followed by P. lilacinum (0.169%), T. harzianum (0.157%), P. fluoroscens (0.146%), P. clamydosporia (0.138%), neem cake (0.134%) and carbofuran 3G (0.106%). The minimum sulphur content was recorded in the control (0.066%) (Table 4).

Micronutrients

The zinc content in mulberry leaves was found to be consistent in all the treatments before the imposition of treatments (initial). The intermediary (60 DAT) observations reveal that the maximum zinc content was recorded in Nemahari (18.90 5). The iron content in mulberry leaves was found to be invariable among the treatments before the imposition of treatments (initial). The intermediary (60 DAT) observations reveal that the maximum iron content was recorded in Nemahari (208.75 ppm) on par with T. viride (205. 5).

Discussion

The moisture content varied considerably during the observation intervals but was consistent among the treatments. The bioagents might have prevented the nematodes from entering and blocking the conducting tissues which increased the total chlorophyll content, total carbohydrates, total proteins, crude fibre, crude protein, ash content and the moisture retention capacity (6 hours after harvest) in the mulberry leaves. The influence of bioagents on the elemental composition of mulberry leaves is highly dependent on the availability of nutrients to the plant system without any hindrance from the root zone or the rhizosphere. The fixed form of nutrients might have converted to exchangeable or available forms due to the action of bioagents while the standard and untreated check without any catalysers resulted in reduced elemental composition.

The enhanced leaf yield and other biochemical and elemental parameters observed in this study can be attributed to the production of growth-promoting substances by bioagents or the generation of toxic metabolites that inhibit nematodes and suppress other deleterious microorganisms. The findings of this investigation corroborate the results reported by Sobitha Devi and Datta (2002) [21] and Dube and Smart (1987) [6] , who observed significant increases in growth and yield of tomato, soybean, tobacco and capsicum in pot and field experiments following inoculation with Trichoderma spp. and

Pseudomonas spp. The influence of Trichoderma spp. could be attributed to the production of enzyme chitinase which might have caused hindrance to the infestation by nematodes, thereby affecting the quality and yield attributes of mulberry (Narasimhamurthy, 2010) [15] .

Acknowledgement

AcknowledgmentsThe authors are thankful to Dr. Naveen DV, Assistant Professor, Department of Soil Science and Agricultural Chemistry, College of Sericulture, Chintamani, UAS-Bangalore for permitting and assisting in carrying out biochemical and elemental analysis in the laboratory. The authors appreciate the farmer's willingness to participate and his cooperation in facilitating the experiment on his farm, which was instrumental in the success of this study.

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