Skip to main content

Boxwood phyllosphere fungal and bacterial communities and their differential responses to film-forming anti-desiccants



Anti-desiccant is a class of agrochemicals widely used to protect plants from water stresses, rapid temperature variations, heat and sunburn, frost and freeze damages, transplant shock, and pathogen and pest attack. Although anti-desiccants are generally considered non-toxic to organisms, it is unclear whether they may impact the phyllosphere microbial communities. In this study, three film-forming anti-desiccant products, TransFilm, Vapor Gard, and Wilt-Pruf were applied to the canopy of two boxwood cultivars ‘Vardar Valley’ and ‘Justin Brouwers’ on April 13 and August 26, 2021. Shoot samples were collected from boxwood plants treated with each of the three products, as well as nontreated control on June 16, August 26 (before the second treatment), and October 18. Microbial and plant genomic DNA was isolated together and 16S rRNA gene and the extended internal transcribed spacer regions were amplified with PCR and sequenced on a Nanopore MinION platform for bacterial and fungal identification.


Bacterial communities were more diverse than fungal communities. At the phylum level, the boxwood phyllosphere was dominated by Proteobacteria and Ascomycota; at the genus level, Methylobacterium and Shiraia were the most abundant bacteria and fungi, respectively. Among the three film-forming anti-desiccants, Vapor Gard and Wilt-Pruf had more impact than TransFilm on the microbial communities. Specifically, broader impacts were observed on fungal than bacterial community composition and structure, with most affected fungi being suppressed while bacteria promoted.


This study addressed several major knowledge gaps regarding boxwood phyllosphere microbiota and the impact of anti-desiccants on plant microbiome. We identified diverse microbial communities of boxwood, a major evergreen woody crop and an iconic landscape plant. We also found differential effects of three film-forming anti-desiccants on the composition and structure of bacterial and fungal communities. These findings advanced our understanding of the associated microbiome of this landmark plant, enabling growers to fully utilize the potentials of microbiome and three anti-desiccants in improving boxwood health and productivity.

Peer Review reports


Plant phyllosphere is colonized by numerous bacteria and fungi, with some performing important functions linked to plant health and productivity. These functions range from mitigating environmental stresses to suppressing plant pathogens, including antagonizing plant pathogens [1], inducing plant systemic resistance [2], improving nutrient acquisition [3, 4], and synthesizing plant growth hormones [5]. Knowing how biotic and abiotic factors may impact phyllosphere microbial communities is fundamental for leveraging beneficial microbes to crop health and productivity.

Boxwood is a widely cultivated ornamental evergreen crop with societal and economic importance [6, 7], and the understanding of its phyllosphere microbial communities has recently improved. Kong et al. [8] showed that the foliar culturable bacterial and fungal endophytes of English boxwood (Buxus sempervirens ‘Suffruticosa’) were associated with the cultivar’s differential tolerance to the boxwood blight disease. They also isolated a Burkholderia SSG strain from boxwood leaf tissues and showed its beneficial activities in nutrient acquisition [3] and pathogen antagonism [9]. Recently, we revealed diverse bacterial communities on the boxwood shoot surface and in the internal tissue and found that canopy cover sprayed with contact and systemic fungicides had a broad and strong impact on the epiphytic bacterial communities [10]. These studies have advanced our knowledge of the biology of boxwood and also highlighted the importance of evaluating other agrochemicals for their potential impacts on the boxwood phyllosphere microbiota—an essential step to establishing a systems approach to utilize the full potential of microbiomes for better boxwood management.

Anti-desiccants (also known as anti-transpirants) are a class of agrochemicals broadly used in agriculture and horticulture to protect plants from drought stress [11, 12], sun burn [13, 14], transplant shock [15, 16], winter injury [17, 18], and plant diseases [19,20,21]. As classified according to their modes of action, three types of anti-desiccants are available on the market: (1) film-forming; (2) reflective; and (3) metabolic [22]. Among them, film-forming anti-desiccant is the most commonly applied. The mechanism of film-forming anti-desiccants is to establish a thin polymeric barrier on the leaf or plant surface to reduce plant water loss during transpiration [22]. A common active ingredient of the film-forming anti-desiccants is di-1-p-menthene (also known as pinolene), a terpenic polymer derived from conifer resin and can be emulsified with water [23]. After spraying, it generates a glossy, flexible, and transparent coating that is impermeable to water vapors [24] and also reduces gas exchange [25], resulting in a decreased net photosynthesis [26]. However, film-forming anti-desiccants are generally considered to be non-toxic to plants [27] and other organisms [28], and can persist in effectiveness for months [29].

Unlike pesticides, the impact of anti-desiccants on plant microbiomes remains unknown. While most studies have focused on the changes in plant physiochemistry [11, 30, 31], growth [26], and yields [25] as affected by anti-desiccants, several others have explored the role that film-forming polymers may play in disease management. Elad et al. [32] showed that di-p-methene containing products Vapor Gard and Wilt-Pruf reduced the incidence of powdery mildew up to 82% and 55% in pot grown cucumber. Similarly, Haggag [19] demonstrated suppressing effects of several film-forming anti-desiccants on cucumber downy mildew in a greenhouse study. Most importantly, he provided electron microscopic evidence to show that kaolin anti-desiccant inhibited spore germination and disrupted the sporangia formation of the casual pathogen Pseudoperonospora cubensis [19]. The controlling effect of anti-desiccants on many other foliar diseases was also reported for various plants [20, 33,34,35]. These studies together imply that the film-forming anti-desiccants may direct or indirectly act against fungi with unclear mechanisms. However, it is still unknown whether and to what degree film-forming anti-desiccants may impact plant microbial communities, particularly those colonizing the phyllosphere. Answering these questions is important for growers to properly use this class of agrochemicals for crop health and productivity while maximizing the benefits of the plant microbiomes.

Film-forming anti-desiccants are frequently applied to care for boxwood overwintering, summer drought, and transplant shocks in landscape and nurseries. The objectives of this study were to characterize the phyllosphere microbial communities and investigate how film-forming anti-desiccants may impact the microbiota. Three anti-desiccant products were applied to the canopy of two boxwood cultivars ‘Vardar Valley’ and ‘Justin Brouwers’, and boxwood shoots were taken two and four months after treatments. Microbial and plant genomic DNA was extracted, and the full length 16 S ribosomal RNA (rRNA) gene and the extended internal transcribed spacer (ITS) regions were amplified, and resultant amplicons were sequenced on a Nanopore MinION® platform for bacterial and fungal identification.


Nanopore sequencing summary

Nanopore MinION sequencing generated a total of 25,391,330 raw reads, with 13,942,987 and 11,448,343 reads for 16 S rRNA and ITS amplicons, respectively. After removing boxwood chloroplast sequences and filtering out samples less than 1,000 reads and operational taxonomic units (OTUs) less than 10 reads, 67,693 and 5,667,266 sequences were retained for 16 S rRNA and ITS amplicons, respectively. Average sequencing coverage was 90.0% for 16 S rRNA (n = 96) and 99.9% for ITS amplicons (n = 96). Rarefaction curves based on the clean reads suggested sequencing for ITS reached near-plateau but the depth of 16 S rRNA was comparably shallow due to the removal of 13,298,594 chloroplast sequences (Fig. S1).

Taxonomic compositions of phyllosphere microbial communities

For the total of 96 samples, the 16 S rRNA reads were assigned to 327 genera, 182 families, 105 orders, 49 classes, and 24 phyla for bacterial communities; the ITS reads were assigned to 328 genera, 198 families, 89 orders, 27 classes, and 2 phyla for fungal communities.

Bacterial communities were diverse with slight variation in relative abundance between the two cultivars. At the phylum level, Proteobacteria dominated the phyllosphere bacterial communities of both cultivars with relative abundance over 65%, followed by Bacteroidetes, Cyanobacteria, Firmicutes, Fusobacteria, and Actinobacteria (Fig. 1a and b). Between the two cultivars, the relative abundance of Bacteroidetes, Cyanobacteria, and Actinobacteria were 3.5–0.1% higher in cultivar ‘Justin Brouwers’ while Firmicutes and Fusobacteria were 0.7% and 0.1% higher in ‘Vardar Valley’. Notably, the relative abundance of Acidobacteria was 2.3% in ‘Vardar Valley’ but less than 1.0% in ‘Justin Brouwers’. At the genus level, Methylobacterium, Brevundimonas, Myroides, Stenotrophomonas, Bacillus, Lichenihabitans, Nostoc, Sphingomonas, Aureimonas, Gloeothece, Burkholderia, Massilia, Nitrosomonas, and Synechococcus were the predominant genera. Of these genera, the relative abundance of Brevundimonas, Bacillus, Lichenihabitans, and Massilia were 0.5–1.9% higher in the ‘Vardar Valley’ than ‘Justin Brouwers’ (Fig. 1e). In contrast, the relative abundance of Methylobacterium, Stenotrophomonas, Sphingomonas, and Aureimonas were 0.6–4.0% higher in the ‘Justin Brouwers’ than ‘Vardar Valley’.

Fig. 1
figure 1

Pie charts showing bacterial (a and b) and fungal (c and d) phylum compositions in ‘Vardar Valley’ and ‘Justin Brouwers’ boxwood shoots. Heatmap showing the relative abundance of the top 15 predominant bacterial (e) and fungal genera (f) in the boxwood cultivars

Fungal communities were less diverse than the bacterial communities and dominated by only two phyla: Ascomycota and Basidiomycota. Particularly, Ascomycota dominated the boxwood phyllosphere with relative abundance over 92.0% in both cultivars (Fig. 1c and d). On the other hand, the relative abundance of Basidiomycota was 7.4% in ‘Vardar Valley’, about 4.0% higher than that of ‘Justin Brouwers’. At the genus level, Shiraia, Alternariaster, Cladosporium, Ramularia, Alternaria, Meira, Leptosphaeria, Pseudopithomyces, Aureobasidium, Acidomyces, Acidiella, Pleosporales, Pseudolasiobolus, Pragmopora, and an unknown genus from phylum Ascomycota were the predominant fungi (Fig. 1f). Of these genera, the relative abundance of Shiraia, Meira, Acidomyces, and Pseudolasiobolus were 0.9–11.1% greater in ‘Vardar Valley’ than in ‘Justin Brouwers’, while the relative abundance of Alternariaster, Cladosporium, Ramularia, Alternaria, Leptosphaeria, Pseudopithomyces, Aureobasidium, and Pragmopora were 0.5–3.7% higher in ‘Justin Brouwers’ than in ‘Vardar Valley’.

The bacterial and fungal genera detected were rather consistent across three sampling times, although the total number of genera seen at each time differed slightly. For example, 296, 280, and 301 bacterial genera were identified from ‘Vardar Valley’ boxwood collected in June, August, and October, respectively, with 250 genera consistently observed across all three months (Fig. S2a). Likewise, 299, 302, and 299 bacterial genera were detected from ‘Justin Brouwers’ boxwood in June, August, and October, respectively, with 259 being seen consistently across all three months (Fig. S2b). For fungal communities, 274, 286, and 312 genera were identified from ‘Vardar Valley’ boxwood in June, August, and October, respectively, with 243 genera consistently present across all three months (Fig. S2c). Similarly, 273, 269, and 280 genera were identified from ‘Justin Brouwers’ boxwood in June, August, and October, respectively, with 222 genera present in all three months (Fig. S2d).

Community compositional changes in response to anti-desiccant treatments

Overall, anti-desiccants had more pronounced effects on the fungal than bacterial communities. Analysis of Compositional of Microbiomes with Bias Correction (ANCOM-BC) identified 96 fungal and 62 bacterial genera differentially abundant when comparing each anti-desiccant to the nontreated control across the two cultivars and the three sampling seasons.

The effect of anti-desiccant was largely suppressive on fungal communities. ANCOM-BC identified 59 and 37 fungal genera with differential abundance for ‘Vardar Valley’ and ‘Justin Brouwers’, respectively, with 54.2% and 67.6% of them were suppressed with anti-desiccants compared to the nontreated controls (Fig. 2a).

Fig. 2
figure 2

(a) Barplot showing the number of significantly (Adjusted P value < 0.05) enriched and suppressed fungal genera in boxwood shoots by anti-desiccant treatment when compared to the nontreated control by cultivar and sampling month. The number was based on the sum of all replicates and the total number of genera is indicated in the parenthesis for each comparison. (b) Venn diagram showing the number of differentially abundant fungal genera in boxwood shoots unique to or shared among anti-desiccants by cultivar and sampling month

Vapor Gard and Wilt-Pruf had broader and stronger impact on fungal communities than TransFilm. Specifically, Vapor Gard and Wilt-Pruf each affected more fungal genera than TransFilm in both cultivars (Fig. 2b). For example, Vapor Gard and Wilt-Pruf impacted 20 and 24 genera, respectively, while TransFilm only affected 11 genera in ‘Vardar Valley’ boxwood (Fig. 3). Likewise, Vapor Gard and Wilt-Pruf impacted 9 and 16 genera, respectively, while TransFilm only affected 4 genera in ‘Justin Brouwers’ boxwood (Fig. 3). These impacted fungi included several predominant genera: Pseudolasiobolus, Pragmopora, Acidiella, Acidomyces, Ramularia, Alternaria, Cladosporium, Aureobasidium, and Meira (Fig. 3). Among them, Pseudolasiobolus, Pragmopora, Acidiella, Acidomyces, and Ramularia appeared to be negatively impacted by anti-desiccant, while Alternaria, Cladosporium, Aureobasidium, and Meira were positively affected by anti-desiccant.

Fig. 3
figure 3

Barplot showing the significant (Adjusted P value < 0.05) fungal genera with log fold abundance change ≥ 0.5 or ≤ -0.5. Taxa that are written in red are the predominant genera

Fungal communities were more responsive to anti-desiccant treatments two months after application than four months later. For both cultivars, ANCOM-BC identified 28 and 25 differentially abundant fungal genera in June and October, two months post-treatment, while 13 genera differed in August, four months after the first treatment in April (Fig. 2b). Additionally, 26 and 21 of the affected fungal genera in June and October had a log fold change of abundance ≥ 0.5 or ≤ -0.5, while it was only 7 genera in August (Fig. 3).

Unlike fungal communities, bacterial communities were mostly promoted by the selected anti-desiccants. Specifically, TransFilm and Wilt-Pruf promoted 14 bacterial genera in cultivar ‘Vardar Valley’ in June (Fig. 4a). Of these promoted genera, ten were solely promoted by TransFilm and two by Wilt-Pruf, while only one genus was promoted by both TransFilm and Wilt-Pruf (Fig. 4b). These promoted genera included several predominant bacteria: Massilia, Lichenihabitans, Synechococcus, Nitrosomonas, and Sneathia (Fig. 4d). Similarly, TransFilm, Vapor Gard, and Wilt-Pruf respectively enriched 19, 7, and 19 genera in cultivar ‘Justin Brouwers’ in October, with TransFilm suppressing only one genus. Of the impacted genera, seven were impacted by all three anti-desiccants and 11 were affected by TransFilm and Wilt-Pruf. Only two were solely impacted by TransFilm and one by Wilt-Pruf (Fig. 4c). The affected bacterial genera with a log fold change of abundance ≥ 0.5 or ≤ -0.5 were Enterococcus, Elizabethkingia, and Burkholderia (Fig. 4d); and Burkholderia, the only predominant bacterial genus, was suppressed by TransFilm. Notably, none of the genera were affected by the three anti-desiccants in August and October in ‘Vardar Valley’ and in June in ‘Justin Brouwers’.

Fig. 4
figure 4

(a) Barplot showing the number of significantly (Adjusted P value < 0.05) enriched and suppressed bacterial genera in boxwood shoots by anti-desiccant treatment when compared to the nontreated control. The number was based on the sum of all replicates and the total number of genera is indicated in the parenthesis for each comparison. (b and c) Venn diagram showing the number of differentially abundant bacterial genera unique to or shared among anti-desiccants for the cultivar ‘Vardar Valley’ in June and ‘Justin Brouwers’ in October. (d) Barplot showing the significant bacteria genera with log fold abundance change ≥ 0.5 or ≤ -0.5. The predominant genera are written in red. Only sampling months or anti-desiccant treatments with significant impact were included

Changes in community structure responding to anti-desiccant treatments

Anti-desiccant altered fungal community structure in ‘Vardar Valley’ but not ‘Justin Brouwers’ boxwood shoots. Specifically, permutational multivariate analysis of variance analysis (PERMANOVA) shows that anti-desiccants accounted for 41.7% (P = 0.0010) and 29.8% (P = 0.0040) of the total variation in fungal community structure in ‘Vardar Valley’ in June and October, two months after the first and second treatments, respectively (Fig. 5). Particularly, Vapor Gard and Wilt-Pruf were the main contributors driving the differences, evidenced by a clear separation of them and the nontreated controls in the first dimension of the Principal Coordinates Analysis (PCoA) ordinations (i.e., x-axis of the PCoA plot) (Fig. 5). Anti-desiccant impact on the fungal community structure of ‘Justin Brouwers’ was insignificant. Likewise, anti-desiccant impact on bacterial community structure was insignificant.

Fig. 5
figure 5

Fungal community structure of two boxwood cultivars as affected by anti-desiccant treatments in the three sampling months. Anti-desiccant effect as analyzed by PERMANOVA is shown in the figure. Boxplot on x- and y- side demonstrates the variation of the community structure that explained by anti-desiccant in the first and second dimension, respectively


To our best knowledge, this is the first study evaluating the impact of anti-desiccant on the phyllosphere microorganisms at the whole community level. We discovered a wealth of fungal genera known as pathogens and saprophytes dominating the boxwood shoots. We also found that the predominant bacteria included many genera that have species and strains known to promote plant growth and health, agreeing with our previous study [10]. Foliar application of three film-forming anti-desiccants had more marked impact on fungi than bacteria in both community composition and structure, especially with Vapor Gard and Wilt-Pruf.

Diverse bacteria and fungi were identified from the two boxwood cultivars, extending our understanding of microbial assemblies in boxwood phyllosphere. Boxwood shoots were dominated by Proteobacteria at the phylum level and Methylobacterium at the genus level, consistent with our previous observation [10]. Many predominant bacterial genera, including Methylobacterium, Stenotrophomonas, Bacillus, Sphingomonas, and Burkholderia, have species and strains known to promote plant growth and health. For example, some Methylobacterium inoculants increased rice yield under the field condition [36] and Burkholderia SSG, an endophytic isolate of boxwood leaves promoted boxwood growth from 37.3 to 76.1% in 10 months [3]. These bacteria may play a pivotal role in the low-maintenance nature of boxwood. On the other hand, the dominant fungi were largely from the phylum Ascomycota, including Shiraia, Alternariaster, Cladosporium, Ramularia, and Alternaria. These fungi contain many species known as plant pathogens and saprophytes. For example, Shiraia-like fungi have been isolated from bamboo tissues as an endophytic parasite [37]. Detection of Shiraia as a dominant genus in boxwood is yet to be confirmed through isolation. Other predominant genera, such as Alternaria and Cladosporium were also isolated abundantly in other boxwood species and cultivars of the same species (i.e., B. sempervirens), particularly in the diseased boxwood plants [38,39,40]. Although these fungi do not directly cause any major boxwood disease, they may play a role in mediating plant defense reactions and accelerating plant senescence [41,42,43,44], making boxwood plants vulnerable to some pathogens [38, 40]. These results highlight the importance of understanding how phyllosphere fungal communities may have contributed to the Western North Carolina region as a hotspot for multiple boxwood diseases.

This study showed that the anti-desiccant treatments had differential impacts on the phyllosphere fungal and bacterial communities. Although the polymer ingredients are considered to be non-toxic to organisms [28], ANCOMBC showed that they impacted 96 fungal genera and 62 bacterial genera. Specifically, 54.2–67.6% of these fungi were suppressed by anti-desiccant treatments, while over 98.0% of the impacted bacteria were promoted. The mechanisms by which these polymer compounds interacted with these fungal and bacterial genera are unclear at this time. Polymers coating could alter fungal spore adhesion to plant surfaces and affect gemination of the spore [45], in addition to providing a physical barrier that prevents fungal direct contacts. Sutherland and Walters [46] also reported three anti-desiccants changed the morphology of the appressoria and haustoria in Blumeria graminis. Some anti-desiccants also suppressed powdery mildew [32, 46], grey mold [33], leaf rust [47], and Guignardia leaf blotch [20]. Compared with fungi, bacteria on the plant surface can produce biofilm, a matrix of extracellular polymeric substances to provide adequate protection and mitigate environmental stresses [48,49,50]. Importantly, the coating layer produced by the film-forming anti-desiccants increases water availability without limiting nutrients leached from plants [27], this may enhance bacterial survival and other activities on the plant surface or inside the tissue [51, 52]. In this study, Vapor Gard (96% di-1-p-menthene) and Wilt-Pruf (25% di-1-p-menthene) had more pronounced effect in altering fungal community composition and structure than TransFilm (8.2% polymeric terpenes and 9.1% oxidized polyethylene), this may be due to the polymer concentration is much lower in TransFilm than the other two products. What caused these differences among the three anti-desiccants is yet to be elucidated in future studies. Additionally, the least changes were observed in the fungal communities in August, four months after anti-desiccant treatments, compared to the broad impacts at two months after treatment, indicating the effect of anti-desiccant may have faded with time. This was not unexpected due to the polymer coating would be naturally degraded, indicated by the recommended application intervals.


This study characterized the shoot bacterial and fungal communities of ‘Vardar Valley’ and ‘Justin Brouwers’ boxwood and assessed the effect of three film-forming anti-desiccants on these microbial communities. We found that the boxwood phyllosphere was a hub for a wealth of fungal genera that are known as pathogens and saprophytes, but the identified bacterial communities included many genera having species and strains known to benefit plant growth and health. The abundance of several predominant fungi varied more between the two cultivars than that of the bacterial communities, this could be attributed to different plant age and size, cultural practices and microclimate. Foliar application of three film-forming anti-desiccants altered both fungal and bacterial community compositions, but the effect was more marked and suppressive on fungi than bacteria. These results advanced our understanding of the associated microbiome for this landmark plant and the impacts of film-forming anti-desiccants on plant microbiomes. This study also provides a good example for examining other anti-desiccant products for their potential effects on crop microbiomes, enabling growers to utilize the full benefits of anti-desiccant action while minimizing their negative impact on the beneficial microbes.


Study sites and boxwood cultivars

This study was done in 2021 at two sites in Western North Carolina; and these two sites were about 35 km apart. A portion of 5-year old plantings of Buxus sempervirens ‘Vardar Valley’ on a commercial farm were used at site (1) Similarly, 3-year old B. sempervirens ‘Justin Brouwers’ were grown at site (2) At the time of the study, ‘Vardar Valley’ plants were about 60 cm in height and 40 to 50 cm in width, and ‘Justin Brouwers’ plants were about 20 cm in height and 12 to 18 cm in width. Both cultivars are slow-growing boxwood plants, at a rate about 2.5 to 7.6 cm annually. Plantings at both sites had not been treated with any anti-desiccants, insecticides, or fungicides before the study. At site 1, fertilizer was applied in early April and two herbicides Roundup® (Bayer AG, Leverkusen, Germany) and Goal® (Nutrichem Co., Ltd., Beijing, China) were sprayed at label rates in late May to manage weed. However, neither fertilizer nor herbicides were used at site 2. Daily temperature and precipitation records from April to October were retrieved from Iowa Environmental Mesonet (, accessed on 10/5/2022) using the closest weather stations EKNN7 (network NC_COOP) and RAVN7 (network NC_DCP) for sites 1 and 2, respectively (Table S1).

Anti-desiccants treatments and sample collection

Three anti-desiccant products TransFilm®, Vapor Gard®, and Wilt-Pruf ® were selected for this study (Table 1). TransFilm contains 8.2% polymeric terpenes and 9.1% oxidized polyethylene. Vapor Gard contains 96% Pinolene (di-1-p-menthene), a terpenic polymer. Wilt-Pruf contains 25% Pinolene. These polymers are classified as film-forming anti-desiccants, as they produce a thin, flexible, and transparent physical layer on the leaf surface. The film hinders the loss of water from the plant but permits diffusion of carbon dioxide [22]. According to the manufacturer, TransFilm also contains diethanolamine, a surfactant compound toxic to microorganisms with an EC50 value of 73 mg/L. The recommended application intervals are 3 to 4 months for TransFilm and Wilt-Pruf, and 3 to 6 months for Vapor Gard (Table 1). Anti-desiccants were freshly prepared following label instructions: 75 ml, 48 ml, and 150 ml of TransFilm, Vapor Gard, and Wilt-Pruf were respectively mixed with 925 ml, 952 ml, and 850 ml of water to prepare one liter of emulsified solution for each product.

Table 1 Emulsified anti-desiccant products included in this study

A randomized complete block design with four replicates was used to include one plant for each of the three anti-desiccant treatments and one nontreated control in a block. Plants were spaced about 90 to 120 cm apart. The first application of anti-desiccants to boxwood canopy was done on April 13,2021, and the second application was conducted on August 26, 2021, using a customized boom sprayer driven by 4-gallon CO2 compression tank for improved coverage on all sides of the boxwood shrubs.

Shoot samples were taken on June 16 and October 18, two months after the application. Another sampling took place on August 26, 4.5 months after the first application (or right before the second application). At the time of sampling, there was no sign of phytotoxicity on any boxwood plants caused by the anti-desiccant. Ten to twelve 5- to 8-cm long boxwood shoots without any disease symptom were randomly selected and taken at the top and middle sections of each plant using a hand pruner (FELCO, Seattle, WA, USA). The pruner was sterilized with 75% alcohol and wiped dry using fresh paper towels between plants. The shoots from each plant were collected into a new Ziploc® (Bay City, MI, USA) bag and then placed in a cooler containing ice packs. Samples were transported to the laboratory at Virginia Beach, VA the next day and stored at -20 °C.

Sample processing, and DNA extraction

From the stem tip, five 5- to 8-cm long shoots were arbitrarily selected from each of the total 96 replicate samples. For each shoot, ‘Vardar Valley’ had an average of 20 leaves, and ‘Justin Brouwers’ had an average of 40 leaves. We used liquid nitrogen to homogenize the shoot samples. The extraction of genomic DNA followed Qiagen Plant Mini Kit (Hilden, Germany) with minor modifications. Specifically, approximately 200 mg ground tissue was further homogenized using a MP FastPrep™ 24 (MP Bio, Irvine, CA, USA) at the speed of 4/s for 1 min in a sterilized zirconium bead tube (500 um garnet and 6 mm zirconium, PFMM 500-100-25U, OPS Diagnostics, Lebanon, NJ, USA) prefilled with 400 µl of AP1 buffer. Four µl of RNase A was added and vortexed for 3 s. Nuclease free water was used to dissolve DNA from the MB Spin Column membrane. The DNA was stored at -20°C.

PCR amplification

PCR amplification of full length 16 S rRNA amplicon followed the protocol in our previous study [10]. The extended ITS regions were amplified with the fungal-specific primers NSA3 and NLC2 [53]. DNA samples were first diluted to 10 ng/µl and a 50 µl PCR reaction mixture was prepared to include 1 µl of the primers, 5 µl of 10x Buffer, 4 µl of dNTP, 0.2 µl of the Takara® polymerase (Takara Bio, San Jose, CA, USA), and 1 µl of the template DNA. The PCR thermal conditions for amplifying the 16 S rRNA gene and ITS regions were the same and detailed in the previous study [10]. The Wizard® SV Gel and PCR clean-up system (Promega, WI, USA) was used to clean up the PCR products.

Library preparation and nanopore MinION sequencing

Nanopore SQK-LSK110 ligation kit and EXP-PBC096 barcode kit (Oxford Nanopore Technology, Oxford, UK) were used to prepare multiplexed sequencing libraries. Procedures and thermal conditions followed the corresponding kit protocols. The AMPure XP beads (Beckman Coulter Life Science, Indianapolis, IN, USA) were used for each clean-up step. Sixteen of each barcoded 16 S rRNA and ITS amplicon libraries were pooled with equal molar amount of DNA to a total of 1 µg library for each sequencing run. Library loading followed the Nanopore priming and loading protocol.

Base-calling, chimera removal, and taxonomy assignments

Nanopore live base-calling and chimera removal for 16 S rRNA reads were specified in Li et al. [10]. To removing chimeras in the ITS amplicon sequences, the full “UNITE + INSD” dataset (for fungi, version 8.3) [54] was used and curated [10] using the Quantitative Insights Into Microbial Ecology (QIIME2) [55] and the REference Sequence annotation and CuRatIon Pipeline (RESCRIPt) [56]. The length of the reference sequences was set at a minimum of 1,000 bp. Minimap2 [57] and yacrd [58] were implemented to pick chimera sequences following Cuscó et al. [59]. To assign fungal taxonomy, the chimera free sequences were then aligned to the curated “UNITE + INSD” reference database with Minimap2 and the top alignment was selected [59]. Here, we adopted the terminology ‘Operational Taxonomic Unit’ (OTU) to describe the taxonomy-assigned sequences. Downstream statistical analyses and visualization were carried out with R software (version 4.2.2) [60]. In R, the sequences were further pruned to remove samples with less than 1,000 sequences and OTUs less than 10 sequences across all the samples. Boxwood chloroplasts sequences were also removed from the 16 S rRNA sequences. Sample coverage was checked using the phyloseq_coverage function from the metagMisc package [61].

Statistical analyses

Differential abundance analysis

Analysis of Compositional of Microbiomes with Bias Correction (ANCOM-BC, version 2.0.2) [62] was used with the ancombc2 function to identify differentially abundant bacterial and fungal genera as a function of anti-desiccant for each of the two cultivars and three sampling months. The maximum number of iterations was set to 500 for both iterative MLE (parameter: iter_control) and E-M (parameter: em_control) algorithms. The False Discovery Rate (FDR) [63] was used to correct P values from multiple tests at the significance level of 0.05.

Microbial community structure

Microbial community structure was measured using the Bray-Curtis dissimilarity index [64] for each cultivar across three sampling months. OTU counts were first normalized with Hellinger transformation [65] by implementing the microbiome package (version 1.20.0) [66], and the dissimilarity distance matrix was then calculated using the vegdist function from the vegan package (version 2.6.4) [67]. Principal coordinate analysis (PCoA) was used to ordinate the dissimilarity matrix. Anti-desiccant effect on microbial community structure was tested using the permutational multivariate analysis of variance analysis (PERMANOVA) [67] with 1,000 permutations. The distances among the anti-desiccant treatments on the first and second dimensions were compared using Tukey’s honestly significant difference (HSD) test. Significance level of all tests was set at 0.05.

Data Availability

The based-called and demultiplexed FASTQ reads are deposited at the National Center for Biotechnology Information Sequence Read Archive (NCBI SRA) under BioProject ID: PRJNA852261. R scripts used in this study are publicly available at



Analysis of compositional of microbiomes with bias correction


False discovery rate


Internal transcribed spacer


Operational taxonomy unit


Permutational multivariate analysis of variance analysis


Principal coordinates analysis


Polymerase chain reaction


Quantitative insights into microbial ecology


Ribosomal RNA


Reference sequence annotation and curation pipeline


  1. Li P-D, Zhu Z-R, Zhang Y, Xu J, Wang H, Wang Z, et al. The phyllosphere microbiome shifts toward combating melanose pathogen. Microbiome. 2022;10:56.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kong P, Li X, Gouker F, Hong C. cDNA transcriptome of Arabidopsis reveals various defense priming induced by a broad-spectrum biocontrol agent Burkholderia sp. SSG. Int J Mol Sci. 2022;23:3151.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kong P, Hong C. Endophytic Burkholderia sp. SSG as a potential biofertilizer promoting boxwood growth. PeerJ. 2020;8:e9547.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Fürnkranz M, Wanek W, Richter A, Abell G, Rasche F, Sessitsch A. Nitrogen fixation by phyllosphere bacteria associated with higher plants and their colonizing epiphytes of a tropical lowland rainforest of Costa Rica. ISME J. 2008;2:561–70.

    Article  PubMed  Google Scholar 

  5. Abadi VAJM, Sepehri M, Rahmani HA, Zarei M, Ronaghi A, Taghavi SM, et al. Role of dominant phyllosphere bacteria with plant growth–promoting characteristics on growth and nutrition of maize (Zea mays L). J Soil Sci Plant Nutr. 2020;20:2348–63.

    Article  CAS  Google Scholar 

  6. Daughtrey ML. Boxwood blight: threat to ornamentals. Annu Rev Phytopathol. 2019;57:189–209.

    Article  CAS  PubMed  Google Scholar 

  7. Hall CR, Hong C, Gouker FE, Daughtrey M. Analyzing the structural shifts in U.S. boxwood production due to boxwood blight. J Environ Hortic. 2021;39:91–9.

    Article  Google Scholar 

  8. Kong P, Sharifi M, Bordas A, Hong C. Differential tolerance to Calonectria pseudonaviculata of English boxwood plants associated with the complexity of culturable fungal and bacterial endophyte communities. Plants. 2021;10:2244.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kong P, Richardson P, Hong C. Burkholderia sp. SSG is a broad-spectrum antagonist against plant diseases caused by diverse pathogens. Biol Control. 2020;151:104380.

    Article  CAS  Google Scholar 

  10. Li X, Tseng HT, Hemmings G, Omolehin O, Taylor C, Taylor A, et al. Characterization of boxwood shoot bacterial communities and potential impact from fungicide treatments. Microbiol Spectr. 2023;0:e04163–22.

    Google Scholar 

  11. Faralli M, Grove IG, Hare MC, Alcalde-Barrios A, Williams KS, Corke FMK, et al. Modulation of Brassica napus source–sink physiology through film antitranspirant induced drought tolerance amelioration that is dependent on the stress magnitude. J Agron Crop Sci. 2017;203:360–72.

    Article  CAS  Google Scholar 

  12. AbdAllah AM, Burkey KO, Mashaheet AM. Reduction of plant water consumption through anti-transpirants foliar application in tomato plants (Solanum lycopersicum L). Sci Hortic. 2018;235:373–81.

    Article  CAS  Google Scholar 

  13. Rodriguez J, Anoruo A, Jifon J, Simpson C. Physiological effects of exogenously applied reflectants and anti-transpirants on leaf temperature and fruit sunburn in citrus. Plants. 2019;8:549.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Melgarejo P, Martínez JJ, Hernández F, Martínez-Font R, Barrows P, Erez A. Kaolin treatment to reduce pomegranate sunburn. Sci Hortic. 2004;100:349–53.

    Article  CAS  Google Scholar 

  15. Berkowitz GA, Rabin J. Antitranspirant associated abscisic acid effects on the water relations and yield of transplanted bell peppers. Plant Physiol. 1988;86:329–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nitzsche P, Berkowitz’ GA, Rabin J. Development of a seedling-applied antitranspirant formulation to enhance water status, growth, and yield of transplanted bell pepper. J Am Soc Hortic Sci. 1991;116:405–11.

    Article  CAS  Google Scholar 

  17. Pair JC, Still SM. Antitranspirant effects on leaf water potential and winter injury of Holly. J Am Soc Hortic Sci. 1982;107:9–11.

    Article  Google Scholar 

  18. Sandler HA. Use of an antitranspirant to minimize winter injury on nonflooded cranberry nogs. HortScience. 1998;33:644–6.

    Article  CAS  Google Scholar 

  19. Haggag WM. Application of epidermal coating antitranspirants for controlling cucumber downy mildew in greenhouse. Plant Pathol Bull. 2002;11:69–78.

    Google Scholar 

  20. Percival G, Keary I, Marshall K. The use of film-forming polymers to control guignardia leaf blotch and powdery mildew on Aesculus hippocastanum L. and Quercus robur L. Arboric Urban For. 2006;32:100.

    Article  Google Scholar 

  21. Glenn DM, van der Zwet T, Puterka G, Gundrum P, Brown E. Efficacy of kaolin-based particle films to control apple diseases. Plant Health Prog. 2001;2:4.

    Article  Google Scholar 

  22. Mphande W, Kettlewell PS, Grove IG, Farrell AD. The potential of antitranspirants in drought management of arable crops: a review. Agric Water Manag. 2020;236:106143.

    Article  Google Scholar 

  23. Francini A, Lorenzini G, Nali C. The antitranspirant di-1-p-menthene, a potential chemical protectant of ozone damage to plants. Water Air Soil Pollut. 2011;219:459–72.

    Article  CAS  Google Scholar 

  24. Iriti M, Picchi V, Rossoni M, Gomarasca S, Ludwig N, Gargano M, et al. Chitosan antitranspirant activity is due to abscisic acid-dependent stomatal closure. Environ Exp Bot. 2009;66:493–500.

    Article  CAS  Google Scholar 

  25. Faralli M, Grove IG, Hare MC, Boyle RD, Williams KS, Corke FMK, et al. Canopy application of film antitranspirants over the reproductive phase enhances yield and yield-related physiological traits of water-stressed oilseed rape (Brassica napus). Crop Pasture Sci. 2016;67:751–65.

    Article  CAS  Google Scholar 

  26. Weller SC, Ferree DC. Effect of a pinolene-base antitranspirant on fruit growth, net photosynthesis, transpiration, and shoot growth of ‘Golden Delicious’ apple trees. J Am Soc Hortic Sci. 1978;103:17–9.

    Article  CAS  Google Scholar 

  27. Davenport D, Hagan R, Martin P. Antitranspirants … uses and effects on plant life. Calif Agric. 1969;23:14–6.

    CAS  Google Scholar 

  28. Garrett RH. Effects of film-forming antitranspirants on fish, water quality, and terrestrial insects. Master of Science Thesis. The University of Arizona; 1979.

  29. Martin JD, Link CB. The potential use of antitranspirants in the greenhouse production of Chrysanthemum. J Amer Soc Hort Sci. 1978;103:327–31.

    Article  CAS  Google Scholar 

  30. Fahey D. Di-1-p-menthene reduces grape leaf and bunch transpiration. Aust J Grape Wine Res. 2019;25:134–41.

    Article  CAS  Google Scholar 

  31. Silvestroni O, Lanari V, Lattanzi T, Dottori E, Palliotti A. Effects of anti-transpirant di-1-p-menthene, sprayed post-veraison, on berry ripening of Sangiovese grapevines with different crop loads. Aust J Grape Wine Res. 2020;26:363–71.

    Article  CAS  Google Scholar 

  32. Elad Y, Ziv O, Ayish N, Katan J. The effect of film-forming polymers on powdery mildew of cucumber. Phytoparasitica. 1989;17:279–88.

    Article  Google Scholar 

  33. Elad Y, Ayish N, Katan J. Control of grey mould (Botrytis cinerea) with film-forming polymers. Plant Pathol. 1990;39:249–54.

    Article  CAS  Google Scholar 

  34. Han J. Use of antitranspirant epidermal coatings for plant protection in China. Plant Dis. 1990;74:263.

    Article  Google Scholar 

  35. Hsieh TF, Huang JW. Effect of film-forming polymers on control of lily leaf blight caused by Botrytis elliptica. Eur J Plant Pathol. 1999;105:501–8.

    Article  Google Scholar 

  36. Sanjenbam P, Shivaprasad PV, Agashe D. Impact of phyllosphere Methylobacterium on host rice landraces. Microbiol Spectr. 2022;10:e00810–22.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Morakotkarn D, Kawasaki H, Seki T, Kawasaki H, Okane I, Tanaka K. Taxonomic characterization of Shiraia-like fungi isolated from bamboos in Japan. Mycoscience. 2008;49:258–65.

    Article  CAS  Google Scholar 

  38. Shin S, Kim J-E, Son H. Identification and characterization of fungal pathogens associated with boxwood diseases in the Republic of Korea. Plant Pathol J. 2022;38:304–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Akıllı Şimşek S, Katırcıoğlu YZ, Çakar D, Rigling D, Maden S. Impact of fungal diseases on common box (Buxus sempervirens L.) vegetation in Turkey. Eur J Plant Pathol. 2019;153:1203–20.

    Article  Google Scholar 

  40. Kurzawińska H, Mazur S, Nawrocki J. Microorganisms colonizing the leaves, shoots and roots of boxwood (Buxus sempervirens L). Acta Sci Pol Hortorum Cultus. 2019;18:149–54.

    Article  Google Scholar 

  41. Bertelsen JR, De Neergaard E, Smedegaard-Petersen V. Fungicidal effects of azoxystrobin and epoxiconazole on phyllosphere fungi, senescence and yield of winter wheat. Plant Pathol. 2001;50:190–205.

    Article  CAS  Google Scholar 

  42. Smedegaard-Petersen V, Tolstrup K. The limiting effect of disease resistance on yield. Annu Rev Phytopathol. 1985;23:475–90.

    Article  Google Scholar 

  43. Tolstrup K. Saprophytic fungi in the phyllosphere of barley and their effects on the plants growth and grain yield [Cladosporium herbarum, Cladosporium cladosporioides, Cladosporium macrocarpum, Sporobolomyces roseus, Alternaria alternata]. PhD Thesis. Royal Veterinary & Agricultural University; 1984.

  44. Swartzberg D, Kirshner B, Rav-David D, Elad Y, Granot D. Botrytis cinerea induces senescence and is inhibited by autoregulated expression of the IPT gene. Eur J Plant Pathol. 2008;120:289–97.

    Article  CAS  Google Scholar 

  45. Walters DR. Disguising the leaf surface: the use of leaf coatings for plant disease control. Eur J Plant Pathol. 2006;114:255–60.

    Article  Google Scholar 

  46. Sutherland F, Walters DR. Effect of film-forming polymers on infection of barley with the powdery mildew fungus, Blumeria graminis f. sp. hordei. Eur J Plant Pathol. 2002;108:385–9.

    Article  CAS  Google Scholar 

  47. Zekaria-Oren J, Eyal Z. Effect of film-forming compounds on the development of leaf rust on wheat seedlings. Plant Dis. 1991;75:231–4.

    Article  CAS  Google Scholar 

  48. Danhorn T, Fuqua C. Biofilm formation by plant-associated bacteria. Annu Rev Microbiol. 2007;61:401–22.

    Article  CAS  PubMed  Google Scholar 

  49. Vorholt JA. Microbial life in the phyllosphere. Nat Rev Microbiol. 2012;10:828–40.

    Article  CAS  PubMed  Google Scholar 

  50. Lindow SE, Brandl MT. Microbiology of the phyllosphere. Appl Environ Microbiol. 2003;69:1875–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Grinberg M, Orevi T, Steinberg S, Kashtan N. Bacterial survival in microscopic surface wetness. eLife. 2019;8:e48508.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Aung K, Jiang Y, He SY. The role of water in plant–microbe interactions. Plant J. 2018;93:771–80.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Martin KJ, Rygiewicz PT. Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiol. 2005;5:28.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Abarenkov K, Zirk A, Piirmann T, Pöhönen R, Ivanov F, Nilsson RH et al. Full UNITE + INSD dataset for Fungi. 2021.

  55. Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol. 2019;37:852–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Robeson MS, O’Rourke DR, Kaehler BD, Ziemski M, Dillon MR, Foster JT, et al. RESCRIPt: reproducible sequence taxonomy reference database management. PLOS Comput Biol. 2021;17:e1009581.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Li H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics. 2018;34:3094–100.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Marijon P, Chikhi R, Varré J-S. Yacrd and fpa: upstream tools for long-read genome assembly. Bioinformatics. 2020;36:3894–6.

    Article  CAS  PubMed  Google Scholar 

  59. Cuscó A, Catozzi C, Viñes J, Sanchez A, Francino O. Microbiota profiling with long amplicons using Nanopore sequencing: full-length 16S rRNA gene and the 16S-ITS-23S of the rrn operon. F1000Rsearch. 2019;7:1755.

    Article  Google Scholar 

  60. R Core Team. R: A Language and Environment for Statistical Computing. 2021.

  61. Mikryukov V, metagMisc. Miscellaneous functions for metagenomic analysis. 2022.

  62. Lin H, Peddada SD. Analysis of microbial compositions: a review of normalization and differential abundance analysis. npj Biofilms Microbiomes. 2020;6:1–13.

    Article  Google Scholar 

  63. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Methodol. 1995;57:289–300.

    Google Scholar 

  64. Bray JR, Curtis JT. An ordination of the upland forest communities of Southern Wisconsin. Ecol Monogr. 1957;27:325–49.

    Article  Google Scholar 

  65. Legendre P, Gallagher ED. Ecologically meaningful transformations for ordination of species data. Oecologia. 2001;129:271–80.

    Article  PubMed  Google Scholar 

  66. Lahti L, Shetty S. Tools for microbiome analysis in R. 2017.

  67. Oksanen J, Blanchet FG, Kindt R, Legendre P, Minchin PR, O’Hara B et al. vegan: Community Ecology Package. 2019.

Download references


Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

We greatly appreciated that two growers allowed us to conduct this study on their properties and provided on-site support as needed. Our appreciation is extended to R. Holtz of Virginia Tech for assisting in anti-desiccant treatments at both research sites. Finally, we would like to thank Dr. Jo Anne Crouch and Briana Pecora for providing useful comments on the manuscript.


This study was supported by the USDA National Institute of Food and Agriculture under award number 2020-51181-32135.

Author information

Authors and Affiliations



XL conceptualized the study, conducted sample collection, DNA extraction, PCR, sequencing, bioinformatics and data analyses, validated the data, and wrote the main manuscript text. OO conceptualized the study, conducted the experiment and sample collection, and reviewed and edited the manuscript. GH, HT, and AT conducted the experiment and sample collection, and reviewed and edited the manuscript. CT scouted the research site and conducted the experiment. PK conceptualized the study, conducted the experiment, and reviewed and edited the manuscript. MD, DL, and FG participated in the study conceptualization, and reviewed and edited the manuscript. CH secured the funding and research sites, conceptualized the study, conducted the experiment and sample collection, provided resources, supervised the project, validated the data, and reviewed and edited the manuscript.

Corresponding author

Correspondence to Xiaoping Li.

Ethics declarations

Competing interests

All authors declared no conflicting interest.

Ethics approval and consent to participate

No human, endangered or protected species was collected in this study. Permission was obtained from the collaborating growers to conduct the study, and the sampling of Buxus sempervirens ‘Vardar Valley’ and ‘Justin Brouwers’ plants was in full compliance with local and national guidelines. Growers’ names were not listed per protocol approved by the Institutional Review Board at Virginia Tech.

Consent for publication

Not applicable.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, X., Omolehin, O., Hemmings, G. et al. Boxwood phyllosphere fungal and bacterial communities and their differential responses to film-forming anti-desiccants. BMC Microbiol 23, 219 (2023).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Anti-transpirant
  • Di-1-p-menthene
  • Phytobiomes
  • Boxwood
  • Crop protection
  • Systems approach
  • Nanopore MinION sequencing