Effect of selected pesticides on phyllosphere microbial diversity in mulberry (Morus alba L.)

Introduction

Pesticides are widely employed in agriculture to enhance crop productivity and ensure food security by controlling pests, weeds and pathogens. Despite these benefits, the excessive and unregulated use of pesticides has led to serious environmental concerns posing significant risks to soil health, water resources and non-target organisms, including humans. Among various pesticide groups, organophosphate and organochlorine compounds are of particular concern because of their persistent bioaccumulation and toxicity, which have resulted in their widespread detection in soil, water and crop products (Tudi et al., 2021; Kumar et al., 2025) [24, 11] . The phyllosphere comprising the above-ground plant surface serves as a dynamic ecological niche where microbial communities contribute to broader ecosystem processes like leaf health, degradation of pollutants etc. Further, it harbours diverse microbial communities, predominantly bacteria that play crucial roles in plant health, nutrient cycling and environmental interactions (Bashir et al., 2022; Liu et al., 2020) [2,12] . When pesticides are sprayed in agricultural fields, the phyllosphere microbes are the first to encounter and interact with the chemicals, often initiating the transformation of xenobiotic compounds. Under such environmental stress, phyllosphere bacteria and fungi secrete extracellular enzymes including hydrolases, transferases, isomerases, ligases etc., which mediate key biochemical reactions such as hydrolysis and redox reactions, thereby contributing to pesticide degradation (Kucharska et al., 2020; Chia et al., 2024) [10,5] . Mulberry (Morus spp.) is an inimitable host plant for the silkworm, Bombyx mori L. However, due to it luxuriant and lush green foliage available all through the year attracts several pests and pathogens and hence is often exposed to various chemicals for pest and disease management. Such chemical application can inadvertently modify the natural leafsurface microbiota, potentially affecting leaf quality, ecological stability and ultimately sericulture productivity (Sunil Kumar and Naika, 2019) [20] . Therefore, understanding pesticide-induced microbial shifts is essential for sustainable mulberry cultivation and sericulture.

Microbial degradation plays an increasingly important role in bioremediation of pesticide-contaminated environments. Several bacterial genera, including Bacillus, Pseudomonas, Enterobacter and Arthrobacter have been reported to degrade organophosphate and organochlorine pesticides through enzymatic pathways, converting toxic compounds into less harmful metabolites (Lourthuraj et al., 2021; Ariffin and Rahman., 2020) [14,1] . Previous studies in several crops have documentary evidence of altered phyllosphere microbial composition and suppression of sensitive taxa while supporting stress-tolerant bacterial groups upon pesticide exposure (Di Benedetto et al., 2017) [7] . However, comprehensive investigations on pesticide-induced changes in the mulberry phyllosphere are limited. Diafenthiuron and abamectin were chosen as a test chemical due to their extensive use in mulberry pest management and have novel modes of action. Previous studies (Shwetha et al., 2024; Gagana Sindhu et al., 2025) [19,8] reported their residual behaviour and relative safety to silkworms. However, their influence on mulberry phyllosphere bacterial diversity remains inadequately explored. In this context, an investigation was carried out on impact of pesticide residue on phyllosphere bacteria communities in mulberry plant. The study adopted both morphological and molecular approaches to assess the influence of selected pesticides on diversity of phyllosphere microbial communities.

Material and Methods

The experiments were conducted in a well-established mulberry garden (Var. V-1) at the Department of Sericulture, College of Agriculture, GKVK, Bengaluru. The field is located in the Eastern dry zone (Zone 5) of Karnataka at 12˚58' N latitude and 77˚35' E longitude and an altitude of 930 m above mean sea level.

Experimental details

Mulberry leaves positioned at 3 rd , 5 th and 7 th from apex representing different levels of maturity were sampled and processed separately from 11 treatments corresponding to different pesticides and safety durations post spray as mentioned below:  T 1 -T 3: Diafenthiuron 50 WP @ 1 g/L (at 8, 15, 21 DAS, respectively)  T 4 -T 6: Abamectin 1.9 EC @ 0.75 mL/L (at 8, 15, 21 DAS, respectively)  T 7: Dimethoate 30 EC @ 2 mL/L (20 DAS)  T 8: Cyanopyrafen 30 SC @ 0.5 mL/L (20 DAS)  T 9: Azadirachtin 1 EC @ 1 mL/L (20 DAS)  T 10: Control (water spray)  T 11: Absolute control  DAS-Days After Spray

Collection of Samples and Isolation of Phyllosphere Microbes

Mulberry leaves were collected at 8, 15 and 21 DAS from the experimental plots treated with selected pesticides. Phyllosphere bacteria were isolated using the spread plate technique, followed by quadrant streaking to obtain pure cultures. Leaves were processed by shaking in sterile distilled water and serial dilutions were prepared up to 10⁻⁶ before plating on nutrient agar. Plates were incubated at 28 °C for 24 hours and distinct colonies were purified for further characterization. Culturable microflora were identified using both conventional morphology-based methods and molecular techniques (Thaochan et al., 2009) [23] . Colonies that differed in appearance were considered to represent different bacterial strains, species, or genera. Based on characteristics such as size, shape, pigmentation, elevation, consistency and margin of the isolated colonies, differentiation and pure culture were obtained by subculturing on the same medium.

Molecular identification of phyllosphere bacterial isolates

The total DNA was extracted from bacterial colonies by inoculating a single colony into nutrient broth and incubating at 37 °C for 24 h. A 1.5 ml of culture was centrifuged at 10,000 rpm for 3 min to obtain a pellet that was resuspended in 400 µL sucrose buffer, vortexed, treated with 32 µL lysozyme and incubated at 60 °C for 10 min. Subsequently, 45 µL of 10% SDS and 5 µL proteinase K were added and the mixture was incubated again at 60 °C for 10 min followed by the addition of 240 µL NaCl and 140 µL freshly prepared 10% CTAB and incubation in a water bath for 10 min. To the suspension, a 500 µL chloroform: isoamyl alcohol (24:1) was added, mixed thoroughly and centrifuged at 12,000 rpm for 10 min. The upper aqueous phase was transferred to a new tube and DNA was precipitated by adding 50 µL of 3 M sodium acetate and 300 µL of isopropanol with gentle mixing and overnight incubation at -20 °C, followed by centrifugation at 12,000 rpm for 15 min. The supernatant was discarded and the DNA pellet was thoroughly washed twice with 1 mL of 70% ethanol, air-dried and resuspended in 40 µL TE buffer. The isolated DNA samples were subjected to RNase treatment (2 µL) and incubated at 37 °C for 30 min (Swathi et al., 2015) [21] . The 16S rRNA gene was amplified from bacterial colonies by PCR using universal eubacterial primer pairs 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1495R (5′-ACGGCTACCTTGTTACGACTT-3′). The PCR was carried out in 50 µL reaction volumes containing 1.5 mM of each primer, approximately 15 ng of template DNA, 5 µL Taq buffer and 1.5 µL Taq DNA polymerase. The PCR cycling conditions consisted of an initial denaturation at 95 °C for 1 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 59 °C for 30 s and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min. Later, the PCR products were subjected to agarose gel electrophoresis where 2 µL aliquots of each PCR product were resolved on 1% agarose gel using 10× TAE buffer, visualized under UV transilluminator and photographed using a gel documentation system (Gel Doc 200, Bio-Rad, USA). The PCR products were stained with 0.5 µg mL⁻¹ ethidium bromide. A 1 kb DNA ladder (Promega) was used as a DNA molecular weight marker to determine the size of the amplified fragments (Swathi et al., 2015) [21] .

16S rRNA gene sequence analysis

The nucleotide sequencing of purified PCR fragments was performed following Sanger sequencing method. The DNA sequences corresponding to the 16S rRNA gene obtained from individual bacterial isolates were reverse complemented using BioEdit software and the resulting sequences were analyzed along with reference sequences retrieved from the NCBI (National Center for Biotechnology Information) GeneBank database using bioinformatics software and bacterial strains were identified accordingly (Matsumoto and Sugano, 2013 and Devaiah et al., 2022) [15,6] .

Phylogenetic Analysis

Phylogenetic relationships among the isolates were inferred using Maximum Likelihood (ML) method with the Tamura-Nei model in MEGA-11 software (Tamura and Nei, 1993) [22] . Bootstrap analysis with 1000 replications was performed to evaluate clade confidence. The final tree, constructed from 27 sequences with 1740 positions, grouped isolates into well-supported clades corresponding to the phyla Bacillota and Pseudomonadota.

Plate 1: PCR amplified 16S rRNA product of phyllosphere of mulberry sprayed with selected chemicals

Results and Discussion Isolation and Characterization of bacterial communities

A total of 52 bacterial isolates were obtained from mulberry leaf samples of pesticide-treated and control plots. Morphological characterization based on size, shape, pigmentation, elevation, consistency and margin revealed that most isolates were white to creamish in colour, circular or irregular in shape with flat elevation and opaque appearance. Gram staining showed a strong predominance of Gram-positive bacteria (94.2 %), mainly belonging to the genus Bacillus (Table 1). This dominance suggests that Gram-positive, spore-forming bacteria possess a selective advantage under chemically stressed phyllosphere conditions, as previously reported for pesticide-impacted plant surfaces (Nicholson et al., 2000; Chen et al., 2021) [17,4] . [16,27] .

Diversity of microbial communities exposed to different pesticide concentrations

Microbial diversity varied markedly across different treatments. A high residual concentration of the chemical lowered species richness as evidenced 8 DAS (T 1 and T 4 ) where only a few tolerant Bacillus species survived (Table 3). High residue levels of both the test chemicals might have exerted strong toxic effects on sensitive bacterial taxa. A similar decline in phyllosphere diversity following pesticide application have been reported earlier in various crops (Moulas et al., 2013; Chen et al., 2021) [16,4] . The microbial diversity gradually increased at 15 and 21 DAS (T 2 , T 3 , T 5 and T 6 ) when the pesticide residues declined over time. However, despite partial recovery, the diversity of microbial community did not match that observed in chemical free controls of T 10 and T 11 (Table 3). Rare Bacillus species such as B. megaterium, B. safensis, B. stercoris and B. albus were detected exclusively in control treatments (T 10 and T 11 ), indicating that early chemical exposure may have longlasting effects on recolonization by sensitive taxa reflecting that the rare microbial members are highly susceptible to disturbance and may not readily recover after pesticide stress (Sangiorgio et al., 2022; Zhu et al., 2023) [18,27] .

The dominance of B. thuringiensis across most pesticidetreated samples suggests that certain Bacillus species may proliferate under reduced competition or possess detoxification capabilities that allow them to exploit chemically stressed niches (Jing et al., 2020; Chen et al., 2021) [9,4] . 4). The predominance of Bacillota is attributed to their high tolerance to environmental and chemical stress, largely due to endospore formation, which enables survival under desiccation, UV radiation and pesticide residues unlike most non-spore-forming bacteria (Nicholson et al., 2000) [17] . Functionally, Bacillus spp. contribute to mulberry leaf health through the production of phytohormones, siderophores and antimicrobial compounds improving leaf quality, suppressing pathogens and upon ingestion by B. mori, they enhance gut microbial balance, digestion, immunity and silk productivity (Li et al., 2022) [13] .

Although less abundant, the taxa Pseudomonadota contribute to nitrogen metabolism and vitamin synthesis, indirectly supporting leaf quality and silkworm health.

Overall, the dominance of Bacillota highlights their strong phyllosphere adaptation and pivotal role in linking mulberry leaf health with improved silkworm performance (Zhang et al., 2024) [26] . [17,18] . The study enables deep understanding of pesticide-induced changes in the mulberry phyllosphere by showing that pesticide application reduces microbial diversity and causes a lasting shift toward dominance of stress-tolerant Bacillus species. The sensitive non-spore forming Proteobacteria were confined to chemical free treatments indicating high vulnerability to chemical stress. Although partial recovery of microbial communities occurred with declined pesticide residue levels, the original community structure restored fractionally reflecting persistent ecological effects. Overall, the findings highlight the long-term impact of pesticides on phyllosphere microbial balance and stress the need for judicious pesticide management in mulberry ecosystem for better leaf health and sustainable sericulture productivity.

Phylogenetic Tree Analysis

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