Abstract
Background Phosphite products have a history of use as fertilizers and fungicides. In contrast to conventional fungicides, phosphites work both directly and indirectly by activating plant defense mechanisms and are proven to be effective against Phytophthora root rot and canker diseases. There are numerous phosphite products on the market labeled as fungicides or fertilizers, but comparative studies on their efficacy and duration of impact are scarce.
Methods We compared the efficacy of commercially available phosphite products against foliar infections of Phytophthora nicotianae on Rhododendron spp. The products were labeled as fertilizers or fungicides and formulated as soluble concentrates, granules, or slow-release tablets. In 2 separate trials, preventive applications were made to the root zone of containerized and field grown Rhododendron spp. Induced resistance was assayed by measuring lesion size following inoculation of detached leaves over time.
Results In the containerized greenhouse study, all phosphite products suppressed lesion development starting as early as 1 week post-treatment and suppression was sustained for 8 to 12 weeks, resulting in significantly reduced lesion area compared to inoculated, non-treated controls. In the field trial, Reliant L (fungicide) and Reliant Dry Phite 28G (fertilizer) suppressed cumulative lesion area 3 weeks post-treatment with effects persisting 6 and 8 weeks, respectively. All products suppressed cumulative lesion area at 4 and 6 weeks post-treatment except the phosphite tablet (fertilizer).
Conclusions Whether labeled as a fertilizer or fungicide or formulated as a liquid or granule, soil applications of phosphite products provided prolonged, systemic protection against foliar Phytophthora spp. infections in Rhododendron spp. to varying degrees.
Introduction
Phosphonate products, including potassium phosphite, are known to protect many plants against various Phytophthora diseases, including root disease, stem canker, and foliar/fruit blight (Erwin and Ribeiro 1996; Deliopoulos et al. 2010; Havlin and Schlegel 2021). The mode of action of these materials has been shown to be twofold, with higher concentrations being directly inhibitory to Phytophthora pathogens and lower concentrations inducing resistance responses in the treated plant (Smillie et al. 1989; Guest and Grant 1991; Jackson et al. 2000). Resistance responses include increased lignin and suberin deposition (Pilbeam et al. 2011), stimulation of defense enzymes (Jackson et al. 2000), release of superoxides, localized cell death, and increased deposition of phenolic materials (Daniel et al. 2005; Daniel and Guest 2006). Phosphite fungicides in plants are fully systemic, moving from the application site to all living portions of the plant, with various application methods, including foliar sprays, trunk sprays, trunk injections, and root zone treatments having been shown to suppress Phytophthora disease or colonization of foliar, stem, and root tissues (Jackson et al. 2000; Daniel and Guest 2006; Garbelotto et al. 2007; Garbelotto and Schmidt 2009; Gentile et al. 2009; Deliopoulos et al. 2010; McMahon et al. 2010; Akinsanmi and Drenth 2013; Crane and Shearer 2014; Bellini et al. 2021; Havlin and Schlegel 2021).
Foliar-applied potassium phosphite treatments to manage Phytophthora root diseases are well studied (Jackson et al. 2000; Deliopoulos et al. 2010; Havlin and Schlegel 2021; Solla et al. 2021). Less research exists regarding root-zone applications of potassium phosphite fungicides to suppress aboveground Phytophthora disease, particularly in woody plants. The potential of root-zone applications to suppress above-ground Phytophthora disease is attractive due to reduced drift to non-target species and because both foliar and stem-injected treatments have potential to cause phytotoxicity (Hardy et al. 2001; Akinsanmi and Drenth 2013; Crane and Shearer 2014; Scott et al. 2016).
Phosphite products are often marketed and applied as fertilizers or “biostimulants” for purposes beyond disease suppression. Various literature reviews highlight multiple systems in which horticultural crop productivity and/or yield are improved by phosphite applications (Rickard 2000; Gómez-Merino and Trejo-Téllez 2015; Cataldo et al. 2022), and at least one study has shown a drought-tolerance priming effect of potassium phosphite application (Fleming et al. 2019). However, it has also been posited that the positive effects are more common in field studies where mitigation of root disease is a likely mechanism (Thao and Yamakawa 2009). Several reviews have also concluded that while a small amount of phosphite is slowly converted to phosphate in plants and soil, and thus available as a phosphorus nutrient source, application of phosphite as a phosphorous fertilizer is inappropriate (McDonald et al. 2001; Thao and Yamakawa 2009; Havlin and Schlegel 2021; Manghi et al. 2021), which is why numerous potassium phosphite products are marketed as 0-0-27 potassium fertilizers and not phosphorous fertilizers.
In this study, we focused on the systemic induction of resistance against foliar Phytophthora infection over time, and not on biostimulant, phosphorous, or potassium fertilization effects; these effects have been extensively studied and reported previously (Rickard 2000; McDonald et al. 2001; Thao and Yamakawa 2009; Gómez-Merino and Trejo-Téllez 2015; Havlin and Schlegel 2021; Manghi et al. 2021). We anticipated the induction of resistance to foliar Phytophthora infections to be universal across labels and formulations of potassium phosphite products based on previously reported mechanistic research on the mode of action of potassium phosphite in other host-Phytophthora pathosystems (Smillie et al. 1989; Guest and Grant 1991; Jackson et al. 2000; Daniel and Guest 2006; Pilbeam et al. 2011), as well as research specific to foliar infection by Phytophthora ramorum Werres, De Cock & Man in ‘t Veld in Rhododendron spp. (Nechwatal et al. 2010). Few studies have investigated the comparative effects of different potassium phosphite formulations (Scott et al. 2015; Hua et al. 2022) with the goal of determining if different formulations, labeled as fungicide, fertilizer, or biostimulant, provide equal protection against Phytophthora infection. To our knowledge, this is the first study to compare the effects of liquid and dry formulations of potassium phosphite in a series of detached leaf assays over time where treatment area (root zone) is spatially separated from infection court (foliage).
Materials and Methods
Phosphite Products
Four phosphite products containing the active ingredient mono- and di-potassium salts of phosphorous acid were used in these experiments. These products were all applied to the soil as a drench or broadcast application to containerized (Experiment 1) or established field-grown (Experiment 2) Rhododendron plants according to the manufacturers’ recommendations (Table 1).
Phosphite product manufacturer, formulation, product type, application method, and treatment rates for Experiment 1 and 2. ai (active ingredient).
Experiment 1: Greenhouse Study
Sixteen, 3.8-L (1-gal) containerized rhododendrons (Rhododendron maximum L.) procured from a commercial nursery were potted up to 7.6-L (2-gal) containers using commercially available pine fines soilless media (SiteOne Landscape Supply, Inc., Roswell, GA). All shrubs were grown under glasshouse conditions consisting of natural day light exposure, moderate temperatures, (23 to 26 °C in spring, 27 to 30 °C in summer), and 75% to 80% relative humidity. Plants were drip-irrigated daily with a total daily volume of approximately 1 L of water.
This experiment employed a randomized complete block design with 4 replicates per treatment group. Treatments consisted of Fortiphite (proprietary product equivalent to Polyphosphite-30, Plant Food Company, Inc., Cranbury, NJ, USA), Reliant Dry Phite 28G (Quest Products Corp., Linwood, KS, USA), Reliant L (Quest Products Corp., Linwood, KS, USA), and non-treated controls. On 2022 May 3, 6 weeks after rhododendrons had been repotted into 7.6-L containers, treatments were applied according to the manufacturers’ recommendations (Table 1). Fortiphite was applied as a solution at 113 mL/ft2, Reliant L as a solution at 1.2 L/ft2, and Reliant Dry Phite 28G broadcast onto the soilless media surface at a rate of 29 g/pot then watered in with 1 L of water.
Two fully expanded leaves of the newest growth were collected from each plant at 48 hours post-treatment (hpt), then again at intervals of 1, 2, 4, 5, 6, 8, and 12 weeks post-treatments (wpt) for use in a detached leaf assay. Leaves were placed on moistened paper towels inside a plastic container, abaxial (lower surface) side up. On each leaf, one-half was wounded with a flame-sterilized dissecting needle and the other left non-wounded. Wounding leaves allowed us to examine systemic resistance and bypass any resistance mechanisms related to leaf penetration, a suggested mechanism behind differences in results on wounded and non-wounded tissue in previous detached leaf assays (De Dobbelaere et al. 2010). Two 5-mm agar plugs were harvested with a cork borer from an actively growing (7 day old) P. nicotianae Breda de Haan isolate (AL1054; GenBank Accession PP587332) grown on one-fourth strength potato dextrose agar (PDA)(henceforth referred to as one-fourth PDA). One plug was placed directly on the wound and the other placed on the non-wounded section. Similarly, the other leaf was mock inoculated by collecting 2 5-mm, non-infested agar plugs of one-fourth PDA, and placing 1 plug directly on the wound and the other on the non-wounded half of the leaf. The plastic containers were misted with distilled water, their lids secured, and then stored at ambient laboratory temperature (21 °C) and light conditions. After 5 days, each leaf was photographed with a scale bar and lesions were measured post hoc using APS Assess 2.0 (American Phytopathological Society, St. Paul, MN, USA). To ensure that the lesions were caused by Phytophthora infection, 2 replicates per treatment group were tested at 48 hours using Agdia’s (Elkhart, IN, USA) Phytophthora immunostrip assay (ISK 92601) per the manufacturer’s instructions.
Experiment 2: Field Study
Twenty Rhododendron catawbiense Michx. representing cultivars ‘Grandiflorum’, ‘English Roseum’, ‘Roseum Elegans’, and ‘Roseum Pink’ were selected from an established understory planting in an arboretum collection in Charlotte, NC, USA. The rhododendrons were planted in 2011 or 2012, now averaging approximately 2.5 m tall (SE = 0.1 m) and 2.0 m wide (SE = 0.1 m). Plants received supplemental overhead irrigation once per week with additional precipitation of 26.2 cm over the course of the study. Average daytime temperature was approximately 20 °C (range of 15 to 26 °C).
Treatments were applied on 2023 April 13 and consisted of Fortiphite, Reliant Dry Phite 28G, Reliant L, Phosphite tablets (Doggett Corporation, Lebanon, NJ, USA) and non-treated controls, with 4 replicates per treatment. Due to potential cultivar variation, we purposefully spread treatment replicates for this experiment evenly across cultivars. Fortiphite (n = 4), Reliant Dry Phite 28G (n = 4), and Reliant L (n = 4) were applied to 2 ‘Grandiflorum’ and 1 each of ‘English Roseum’ and ‘Roseum Elegans’. Phosphite tablets were applied to 2 ‘Grandiflorum’ and 2 ‘English Roseum’. Non-treated controls consisted of 2 ‘Grandiflorum’ and 1 each of ‘Roseum Elegans’ and ‘Roseum Pink’. All products were applied per the manufacturers’ rate recommendations to an area equivalent to the dripline of each rhododendron (Table 1). Fortiphite was applied as a solution at 113 mL/ft2 and Reliant L was applied as a solution at 1.2 L/ft2. Reliant Dry Phite 28G was broadcast on the soil surface, and phosphite tablets were buried in the soil, evenly spaced, and then watered in with the equivalent of 2.54 cm of water (2.8 L/ft2). Non-treated controls received only water, in the same amount. All treatments were applied within the dripline of each rhododendron.
Similar to Experiment 1, 2 fully expanded leaves of the newest growth were collected from each plant at 1, 2, 3, 4, 6, 8, and 12 weeks post-treatment (wpt) and used in a detached leaf assay. Leaves were placed on moistened paper towels inside a plastic container, abaxial side up. On each leaf, wounds were made in 2 locations on opposite sides of the midvein with a flame-sterilized dissecting needle. Two 5-mm agar plugs were harvested with a cork borer from an actively growing (7 day old) P. nicotianae isolate (AL1054; GenBank Accession PP587332) grown on one-fourth PDA and placed on the leaf wounds. Similarly, the other leaf was mock inoculated by harvesting 2 5-mm, non-infested, one-fourth PDA plugs and placing on each wounded site. The plastic containers were misted with distilled water, their lids secured, and then stored at ambient laboratory temperature (21 °C) and light conditions. After 5 days, each leaf was photographed with a scale bar and lesions were measured post hoc using APS Assess 2.0.
Statistical Analyses
In Experiment 1 (wounded and non-wounded sites) and Experiment 2 (both wounded sites), cumulative lesion area data were (x + 1) Box-Cox transformed to normalize variance prior to conducting an analysis of variance (ANOVA) across all treatments within each time point independently. Means were separated with Tukey’s HSD when there was a significant (P ≤ 0.05) ANOVA for treatment effect. All statistical analyses were calculated in JMP 16.0 (SAS, Cary, NC, USA).
Results
Experiment 1: Greenhouse Study
Disease suppression, reported as significantly smaller cumulative lesion size of 2 inoculated sites relative to the non-treated inoculated control sites, was first observed at 1 week post-treatment (wpt) for all phosphite products (Figure 1). This suppression was sustained until 6 weeks post-treatment for all products, where lesions were on average 77.9% (1 wpt), 89.1% (2 wpt), 96.1% (4 wpt), 95% (5 wpt), 93% (6 wpt) percent smaller than the non-treated inoculated controls. Eight weeks after treatment, all leaves from treated plants continued the trend of smaller lesion sizes (on average 83% smaller lesions than the nontreated inoculated controls) but were not significantly different due to large variation. However, at 12 weeks after treatment, disease suppression was again significant in rhododendrons treated with Reliant Dry Phite 28G and Fortiphite (on average 89% smaller lesions than non-treated inoculated controls), but not Reliant L (Table 2).
Photograph illustrating treatment effects on suppression of Phytophthora infections in detached leaf assays over time where leaves were inoculated and then incubated for 5 days before photographing (Experiment 1).
Treatment effects on detached wounded Rhododendron spp. leaves from the greenhouse study (Experiment 1), following artificial inoculation with an isolate of Phytophthora nicotianae. hpt (hours post-treatment); wpt (weeks post-treatment).
Experiment 2: Field Study
Disease suppression was first observed in established, field-grown rhododendrons at 2 weeks post-treatment where lesions trended smaller than in the non-treated inoculated controls for rhododendrons treated with Fortiphite, Reliant Dry Phite 28G, and Reliant L (on average 76% smaller lesions than the non-treated inoculated controls), although the effect was not statistically significant due to large plant-to-plant variation and fully mature, hardened-off leaves. At 3 weeks post-treatment, rhododendron leaves had significantly (P ≤ 0.05) smaller lesion size relative to the non-treated inoculated controls for Reliant Dry Phite 28G and Reliant L treatments. Significant (P ≤ 0.05) suppression was observed for all products at 4 and 6 weeks post-treatment (on average lesions averaged 94% and 98% smaller than non-treated inoculated controls, respectively) except the phosphite tablet product. At 8 weeks after treatment, Reliant G was the only treatment that had significantly (P ≤ 0.05) smaller lesion size compared to the non-treated inoculated controls (92%). At 8 weeks, slower lesion development was observed in all groups including the non-treated inoculated controls compared to previous weeks. By 12 weeks post-treatment, there were no differences in lesion size for any of the plants and lesion development was negligible (Table 3).
Treatment effects on detached wounded Rhododendron spp. leaves from the field study (Experiment 2), following artificial inoculation with an isolate of Phytophthora nicotianae. wpt (weeks post-treatment).
Discussion
In these experiments, lesion development caused by Phytophthora nicotianae on detached rhododendron leaves was used as a model system to evaluate the capabilities of different root-zone applied potassium phosphite products to induce systemic resistance in a host and pathogen combination known to result in foliar disease (Erwin and Ribeiro 1996; Donahoo and Lamour 2008; Linderman and Benson 2014). In Experiment 1 (greenhouse), treatments with all products tested resulted in reduced lesion area when detached leaves were inoculated at 1 week post-treatment, but not at 48 hours post-treatment. This significant treatment effect lasted up to week 12 for 2 of the products (Fortiphite and Reliant Dry Phite 28G), although the differences were not significant in week 8 due to high variability. In Experiment 2 (field), a trend toward smaller lesion size was observed 2 weeks post-treatment for all products except the phosphite tablet, but none of treatments differed significantly from the untreated control at P ≤ 0.05. A significant reduced lesion size was evident at 4 and 6 weeks post-treatment for all treatments except the phosphite tablet.
While induced systemic resistance as evidenced by suppressed symptom development in inoculated leaves was demonstrated in both our greenhouse and field studies, the results were notably different between the 2 experiments, which is why the data were not combined in our statistical analyses. No lesions developed in mock inoculated leaves and therefore those data were not presented. In the greenhouse experiment, sizable lesions developed in the non-treated inoculated controls at all time points. In contrast, lesions in the non-treated inoculated controls of the field trial only became notable 2 weeks into the experiment and by week 6 were greatly reduced again. This difference is likely due to the developmental stage of the leaves used in the detached leaf assay. Expanded leaves were harvested from the newest growth at each time point, regardless of leaf maturity at that time. In the greenhouse, new flushes of growth were continuously produced and at no point were inoculated leaves considered fully hardened. In the field trial, leaves sampled in the first 2 weeks were fully hardened growth from the previous season, while in weeks 3 and 4 the leaves were taken from the developing new growth. By week 6, leaves were noticeably more hardened and were likely fully mature by week 8. This is similar to results seen when young and old rhododendron leaves were challenged by P. ramorum in detached leaf assays in the absence of chemical treatment (De Dobbelaere et al. 2010). In that study, De Dobbelaere et al. (2010) also found that detached, wound-inoculated young leaves were more susceptible than older wound-inoculated leaves as evidenced by greater lesion development. Another experiment, although investigating a different infection court, found that lesion development on branch cuttings inoculated with P. ramorum was also significantly influenced by host phenological stage (Dodd et al. 2008).
An aspect of the experiments reported here that has been scantly investigated previously is the evaluation of treatment effects at multiple time points over an extended period. Many experiments have examined the systemic effects of phosphite products with inoculation at a single time point after treatment. For example, Khdiar et al. (2023) inoculated roots 48 hours after foliar treatment, while Crane and Shearer (2014) inoculated detached branches 28 days after stem injection, bark spray, and foliar treatments. Similarly, Jackson et al. (2000) assessed root defense responses and lesion length when roots were challenged with Phytophthora cinnamomi Rands at 0, 2, 5, 8, and 14 days after foliar phosphite treatment. They found that there was some reduction in lesion length at 2 days post-treatment, but the greatest reduction in root lesions occurred when inoculated between 8 and 14 days after treatment. In another experiment where inoculation tests occurred at multiple time points, Rolando et al. (2017) found that foliar applications of potassium phosphite significantly reduced lesion length on inoculated needles of Pinus radiata D. Don at 6 days but not 90 days after treatment when challenged by Phytophthora kernoviae Brasier, Beales, and S. A. Kirk. Conversely, in the same study significant reduction in lesion size was observed at 90 days but not 6 days after treatment when challenged by Phytophthora pluvialis Reeser, W. Sutton, and E. M. Hansen.
This detached leaf-inoculation model system allowed us to compare treatment effects of various potassium phosphite products in a time series; however, it must be noted that effects on other Phytophthora species and isolates and on additional Rhododendron species and cultivars may differ due to variation in pathogen sensitivity to phosphite and its effects (Weiland et al. 2021; Hunter et al. 2022), as well as variation in disease susceptibility across Rhododendron spp. genotypes (Linderman et al. 2006; Crane and Shearer 2014). For example, susceptibility of Rhododendron ssp. genotypes to P. nicotianae varies, as demonstrated by Linderman et al. (2006), who ranked 3 different Rhododendron species (none of which were used in our experiments) from most to least susceptible to P. nicotianae based on detached leaf assays. In another study, 2 of the cultivars used in our field experiment (‘Grandiflora’ and ‘Roseum elegans’) were classified in the second highest susceptibility group to Phytophthora ramorum based on detached leaf assays. In the same study, all the R. catawbiense cultivars and hybrids were classified in the highest or second highest susceptibility group (De Dobbelaere et al. 2010). In addition, it is likely that other factors unrelated to the specific potassium phosphite formulation, host, and pathogen species will influence the lag time before resistance is induced and the duration of that resistance. These factors may include plant size, soil type, soil moisture, irrigation or rainfall frequency, health of treated plant and its root system functionality, and weather conditions.
The efficacy of different application methods of single potassium phosphite formulations has been reported. Garbelotto et al. (2007) investigated the protection against P. ramorum in coast live oak (Quercus agrifolia Neé) provided by trunk sprays, foliar sprays, and trunk injections of the same potassium phosphite formulation, with or without surfactants. Similarly, Crane and Shearer (2014) compared phosphite stem injections, stem sprays with 2 different surfactants, and aerial sprays in their ability to protect 3 different plant species against P. cinnamomi, again with a single potassium phosphite formulation. To our knowledge, this is the first report evaluating the induced resistance effects of 3 (greenhouse) or 4 (field) different potassium phosphite formulations, as well as the first report comparing root-zone applied potassium phosphite products labeled as a fungicide to those labeled as fertilizers against foliar Phytophthora infections.
Both our field and greenhouse experiments showed significant activity in inducing resistance following applications of either granular or liquid formulations, as well as in the fertilizer products (Fortiphite, Reliant Dry Phite 28G) and the fungicide product (Reliant L). This is in agreement with the one published report investigating 2 different potassium phosphite formulations (Scott et al. 2015), where the researchers found equal protection in detached branch inoculation assays afforded by either an injected liquid potassium phosphite formulation or a ‘soluble capsule implant’ when challenged with P. cinnamomi. By contrast, under our research conditions, the potassium phosphite tablet formulation placed in the root zone did not prove effective at any time point in reducing the lesion development on detached, inoculated leaves compared to non-treated inoculated control leaves. This could be due to the low dose of active ingredient being released from the tablet when the leaves are susceptible to infection (i.e., expanding leaves) due to low microbial activity that may not peak until leaves harden off later in the springtime following the infection window.
Conclusions and Significance
To our knowledge, this is the first report comparing different phosphite product formulations (i.e., soluble concentrate, granular, etc.) or product types (fertilizers vs. fungicides). In these experiments, there were few differences between the fertilizer and fungicide products and the liquid and granular products also performed similarly. The slow release phosphite tablets were not effective with the methods employed, possibly due to environmental or edaphic conditions. We demonstrated that preventive soil applications of phosphite products induced systemic resistance in actively growing leaf tissues, and that disease suppression can persist for up to 12 weeks post-application. This information can help landscape professionals, nurserymen, and foresters improve recommendations for type and frequency of phosphite application when developing an integrated pest management program for foliar Phytophthora diseases of Rhododendron spp. and likely other woody plants.
Acknowledgements
We would like to thank the F.A. Bartlett Tree Experts Company and Mr. Robert A. Bartlett, Jr. for supporting this research financially, and Isabel Marez, Amber Stiller, Mara Lind, and Annaliese Sander for assistance in these experiments.
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