This study used a modified low-cost impaction spore trap designed by Chandrasekar (2022). The trap consists of a photovoltaic module, 12 V DC, 300 RPM gear motor, 6,000 mAh Li-ion battery, solar panel, metallic sampling rods, rotating arm, and steel stand (Figure 1).

Sampling rods designed for capturing airborne particles were crafted from 60-mm long and 3 mm-wide stainless steel, following the method outlined by Thiessen et al. (2018) with slight modifications. These rods were sterilized and coated with silicone vacuum grease, and then fixed to the rotary arm of the spore trap for sampling.

The trap was set up at Paddy Breeding Station, Coimbatore, Tamil Nadu, India (Latitude 11.0068 °N, Longitude 79.9242 °E) with an average rainfall of 670 mm per year. Geographically, it is located at an elevation of 426.72 m, with an average annual minimum temperature of 22°C and a maximum temperature of 33°C for the year 2021 (Indian Meteorological Department official website, 2021, https://mausam.imd.gov.in/imd). The paddy variety Co-39, which is susceptible to false smut, was grown in the nursery, and the 20-day-old seedlings were transplanted in the main field with a spacing of 20 × 15 cm. The spore trap was operated continuously from transplanting to the harvesting stage of rice crops from the 45th Standard meteorological week of 2023 to the 6^th Standard meteorological week of 2024. Sampling rods were collected aseptically from the trap using sterile containers on a weekly basis, with two rods collected each week and replaced with sterilized rods freshly coated with grease.

The temporary mounts were prepared by gentle scraping of airborne inocula adhered to the grease of sampling rods collected from the spore trap fixed under field conditions and observed under 40x magnification in a phase contrast microscope for observing false smut conidia. The presence and absence of inocula are mentioned by the + or − sign (Table 1).

DNA was extracted according to the method outlined by Quesada et al. (2018). The air particles adhered to the silicone grease of the sampling rods were scraped off using a sterilized toothpick. The scraped material was transferred to a screw-capped vial containing 5 ml of cetyltrimethylammonium bromide (CTAB) buffer. The vial was vortexed, and 0.5 ml of this mixture was transferred to a 1.5 ml centrifuge tube. The tube was incubated at 65°C for 20 min. Subsequently, an equal volume of phenol, chloroform, and isoamyl alcohol (25:24:1) was added, followed by centrifugation at 12,000 rpm for 10 min. The resulting supernatant was collected, and 600 μl of ice-cold isopropanol was added. This mixture was then incubated overnight at −20°C. The sample was centrifuged after 24 h at 10,000 rpm for 10 min, followed by an ethanol wash. The supernatant was discarded, and the remaining pellet was resuspended in 50 μl of TE buffer for storage.

The extracted DNA was then subjected to a conventional PCR check using species-specific primers US1 and US3 (Table 2) to identify the presence of the target species, following the conditions mentioned by Zhou et al. (2003).

The LAMP reactions mixture was made using 25 μl of LAMP reagents containing 2.5 μl of extracted sample DNA, 2.5 μl of Thermopol buffer (10X), 1.4 mM of each dNTPs, 0.3 mM of both outer primers, 1.5 mM of both inner primers (Table 2), 1.2 M Betaine, 8 mM MgSo_4, 2 U Bst DNA Polymerase (0.08 U/μl), and 120 μM of Hydroxy napthol blue (HNB) dye (Logeshwari et al., 2022). The total reaction mixture, except sample DNA, served as a control. The reaction was performed in an Eppendorf thermal cycler for 60 min at a constant temperature of 65°C. Then, the reaction was halted by thermal denaturation at 80°C for 2 min.

The colorimetric observation was conducted after reaction termination, in which the sky-blue color indicated a positive reaction and the purple color indicated a negative reaction. The quality of amplification was evaluated using 2% gel electrophoresis. The presence of a ladder-like pattern indicated the positive amplification.

The sampling rods were collected at weekly intervals from transplanting to harvest of 45th Standard Meteorological Week (SMW) to 6th SMW of 2023–2024, respectively. The DNA was extracted from each sample rod, as described earlier. The presence of airborne inocula was detected using the LAMP assay (2.4). The field trial was conducted using a randomized block design (RBD) with three treatments and seven replications to evaluate the effectiveness of an inoculum-based fungicidal spray schedule compared to the farmer’s practice. The broad-spectrum fungicides Azoxystrobin 16.7% + Tricyclazole 33.3% @ 0.1% concentration were used. Detailed information on treatment is given in Table 3. To evaluate the relationship between meteorological parameters and false smut disease incidence, field data on both environmental conditions and disease incidence were collected during the cropping season. The weather data were obtained from the automatic weather station (Yuktix Technologies, Bangalore, Karnataka) located at Paddy Breeding Station Coimbatore. The parameters such as maximum temperature (°C), minimum temperature (°C), relative humidity morning (%), relative humidity evening (%), and wind speed were recorded during the cropping season (45th to 6th SMW of 2023–2024). The disease incidence was determined by calculating the percent disease incidence (PDI) based on visual field observations of false smut symptoms on infected rice plants. Weekly disease incidence was recorded for different treatments (T1, T2, and T3). The disease incidence was determined according to Baite et al. (2020) using the following formula:

A Pearson correlation analysis was conducted to assess the relationship between meteorological parameters and disease incidence. Pearson’s correlation coefficient (r) was used to measure the strength and direction of the linear relationships between disease incidence (a dependent variable) and each of the meteorological parameters (independent variables). The correlation analysis was performed using statistical software R (version 4.3.1) with the cor function. The weekly meteorological data (relative humidity, wind speed, temperature, sunshine, and solar radiation) were paired with the corresponding disease incidence data for each week (W45–W06). The results of the correlation were interpreted based on r values: (+1) indicates perfect positive correlation (as one variable increases, the other increases), (−1) indicates perfect negative correlation (as one variable increases, the other decreases), and 0 indicates no correlation.

A graphical representation (Figure 2) showed the trends of meteorological parameters and disease incidence over time. Weekly trends were plotted using ggplot2 in R, where disease incidence was compared against key weather variables. This approach allowed for a comprehensive understanding of how weather conditions influenced the progression of false smut disease in the field over time.

To determine the lowest limit of detection using the LAMP assay, the purified genomic DNA was quantified using Nano-Drop One (Thermo Fisher Scientific, Waltham, MA, United States; Figure 3). Ten-fold serial dilutions were prepared from 10 ng to 1 fg of genomic DNA in nuclease-free water. One microliter of DNA from each dilution was added to the individual LAMP reaction mixture. The LAMP assay was performed following the same conditions and components described above. The DNA extracted from the conidial suspension spiked sampling rods was also detected through LAMP by adding 1 μl of DNA to the reaction mixture from each spiked sample. A negative control was included in each run.

Smut ball samples were collected from the infected plant samples at the paddy breeding station Coimbatore, Tamil Nadu, in 2023. To isolate the pure culture, the smut balls were surface-sterilized with 0.1% sodium hypochlorite and rinsed three times with sterile water. The smut balls were then dried on sterilized filter paper. Using a sterilized needle, the chlamydospores were streaked on the potato sucrose agar plate amended with streptomycin, per the protocol followed by Ladhalakshmi et al. (2012). The plates were incubated at 27°C for 7 days. The pure culture was obtained by repeated subculturing. The 100 ml milky grain broth (165 ml milky grain extract, made up to 1,000 ml with sterilized water) was inoculated with the agar plug from the pure culture, and it was incubated for 8 days at 25°C and 120 rpm. After that, 100 ml of potato sucrose broth (PSB) was added, and the mixture was once more incubated under the same conditions for a further week. Following this step, a cheesecloth was used to filter the culture, and then 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO, USA) was added to the suspension. The conidial suspensions were then pipetted onto rod sets placed in the vial.

To assess the sensitivity of the LAMP assay, the sampling rods were then spiked with a known concentration of serially diluted spore suspensions of U. virens. The conidial concentration was measured using a hemocytometer. Initially, the concentration was approxiamtely 1 × 10⁴ conidia/ml. It was then 10-fold serially diluted to 1 × 10³, 1 × 10², 1 × 10, and 1 conidia and spiked to the sampling rod, respectively; the rod spiked with 0 conidia (only nuclease-free water) served as a control (Villari et al., 2017). Before further processing, the rods were allowed to air dry under laboratory conditions (28 ± 2°C) for 24 h (Figure 3).

U. virens conidia were detected using LAMP assay from the sample collected from an air sampler (spore trap) placed in the trial plot during the cropping period. The spore dispersal was recorded at weekly intervals from the 45th to the 6th SMW of 2023–24 by collecting sampling rods from the spore trap. The microscopic observation of mounts prepared from sample rods revealed the presence of U. virens, Bipolaris oryzae, Pyricularia oryzae, Alternaria, and rice pollens (Supplementary Figure S1). Initially, the U. virens spores were not seen under a microscope. The spores were detected only at 48th SMW both under the microscope and LAMP assay, but in conventional PCR, it is detected only a week before visible symptom expression, i.e., on 50th SMW (Supplementary Figures S2, S3). This implies the higher sensitivity of the LAMP assay compared to conventional PCR to detect the lowest possible inoculum of U. virens. The first visible symptoms were seen only after 3 weeks from the detection of airborne inocula through the microscope, mainly observed at the panicle exertion stage 51^st SMW. The presence and absence of U. virens was confirmed primarily by microscopic observation. Chlamydospores were mostly seen under microspores in the sample collected from the spore trap. Further confirmation was achieved by the development of sky-blue color and ladder-like pattern formation under 2% agarose gel electrophoresis.

The spray schedule was developed by inoculum-based spraying of azoxystrobin and tricyclazole as a single spray @ 0.1% at 50th SMW after booting and before flowering was recorded, with the minimum incidence of false smut (4.48%) with a maximum yield of 6,500 kg/ha. On the other hand, blanket application of fungicides based on farmers’ practices recorded a maximum of 50.67% of false smut incidence with a yield of 3,500 kg/ha. In the untreated control, the incidence of false smut reached the maximum of 58.53% and recorded a minimum yield of 1,500 kg/ha (Table 1; Figure 2). Similarly, an association was found between the frequency of disease and the meteorological parameters that dominated during the cropping season. Specifically, disease incidence increased with relative humidity and wind speed and decreased with temperature, sunshine, and solar radiation (Supplementary Tables S1, S2).

The limit of detection of the U. virens using PCR assay was determined by performing the assay on a tenfold serially diluted purified genomic DNA. The PCR reaction detected the target DNA at a minimum concentration of 10 pg (Figure 4). A total of six independent spore dilution series were used by adding 2.5 μl of DNA derived from sampling rods spiked with 0 to 1 × 10⁴ conidial spores. The PCR assay was able to amplify up to 1 × 10² conidia at 380 bp, while it failed to amplify 1 and 10 conidia per sampling rod.

The limit of detection of the U. virens LAMP assay was determined by performing the assay on a tenfold serially diluted preparation of purified genomic DNA. The results revealed strong sky-blue color development in all tubes containing different DNA concentrations except those containing 10 and 1 fg of DNA. Similarly, the ladder-like pattern was seen at a minimum concentration of 100 fg under 2% agarose gel electrophoresis (Figure 5). The LAMP amplified positive for all the serially diluted conidial spiking except for single conidia. The lowest possible detection of LAMP through spiking assay was about 1 × 10 conidia per sampling rod.

Three operators performed the LAMP assay with blind samples, including U. virens and other species, and a non-template control (Table 4). As a result, all three operators correctly identified U. virens, and there was no cross-reactivity with any other non-target samples.