Polyoxin D is purified from the culture broth of actinomycetes and used as an agricultural fungicide. This compound shows antifungal activity against various plant pathogenic fungi, especially Rhizoctonia solani, by inhibiting germination, hyphal growth, and sporulation of the fungi. Its mode of action is thought to be via the competitive inhibition of chitin synthase, which causes incomplete cell wall formation and swelling in germ tubes, hyphae and septa. This action is unique and results in the suppression of diseases in various crops through fungistatic rather than fungicidal activity. Although the polyoxin complex shows not only fungicidal but also insecticidal activity, polyoxin D has less activity against insects, no negative impacts on the environment and is suitable for organic agriculture. Polyoxin D has the potential to control soil-borne and post-harvest diseases and to inhibit mycotoxin production via a new mode of action.

“Polyoxins”, consisting of 14 ingredients from polyoxin A to L, N, and O, were discovered in 1961 in the culture broth of an ascomycete isolated by Dr. Saburo Suzuki of the Institute of Physical and Chemical Research, RIKEN in Japan, from soil collected from the Aso region of Kumamoto Prefecture in Japan.¹⁾ The ascomycete was identified to be Streptomyces cacaoi Waksman and Henrici (S. cacaoi) var. asoensis Isono.¹⁾ Almost all of the ingredients induce morphological changes such as swelling in plant pathogenic fungi and inhibit the associated diseases. Many researchers have worked to elucidate their biological activity in crop protection using single polyoxin components or mixtures of polyoxins. In particular, polyoxin D zinc salt (MW: 584.756 g/mol; hereinafter referred to as “polyoxin D”) has garnered much research interest due to its unique mode of action, and the scientific literature on polyoxin D, including new findings, has been rapidly accumulating in recent years.

Since the 1960’s, the negative impact of pesticides on humans, livestock, and the environment has been a global issue. A mixture of polyoxins was developed as a very safe agricultural fungicide and was first registered for use in Japan in 1967 for control of rice sheath blight caused by Rhizoctonia solani Kühn (R. solani). Since then, some products containing polyoxin complex or polyoxin D zinc salt as active ingredients were registered for controlling various diseases in fruit trees, vegetables, flowers, and turf in approximately 20 countries. In the US, polyoxin D is regulated as a biochemical active ingredient because it is produced via a fermentation process using a naturally-occurring microbe, and it has non-toxic mode of action to target fungal pathogens.²⁾ Polyoxin D is listed by the National Organic Program as an allowed synthetic active ingredient that is permitted for use in organic farming.³⁾ In addition, the polyoxin D 5% suspension concentrate formulation (5%SC) is listed by the Organic Materials Review Institute (OMRI), USA as a permitted reagent for organic crop production. There is no human or veterinary medicinal use of polyoxin complex or polyoxin D.

As many countries around the world are working to reduce the environmental impacts of agriculture, and as part of this effort are aiming to reduce the use of synthetic chemical pesticides, there is an urgent need to establish alternative pest control methods. Copping and Menn⁴⁾ have introduced polyoxins as one of the microorganisms-derived biopesticides. In this review, we focus on polyoxin D manufactured by fermentation technology and discuss its agricultural advantages based on the current literature as well as unpublished original findings regarding its antifungal activity, mode of action, rain resistance, residual activity in the field, and effects on fungi resistant to other fungicides and biological pesticides. Further, the future development and application of polyoxin D for crop protection and research topics from the perspective of plant pathology and molecular biology are also explored here.

Polyoxins, which were isolated and identified as active substances against fungi in secondary metabolites of actinomycetes, have historically been classified as nucleoside antibiotics.⁵⁾ However, polyoxins are not active against bacteria such as Streptomyces spp., Escherichia coli NIHJ and Bacillus subtilis ATCC6633.⁶⁾ To search for new substances showing higher biological activity than polyoxins, the synthesis of their derivatives and structure–activity relationship studies have been carried out, but only polyoxins have been put to practical use, so far.⁵⁾ In this chapter, we have reviewed the literature related to the basic activity of polyoxin D as an agricultural fungicide and have attempted to derive a scientifically consistent view from those results in spite of different test methods used by researchers. For quantitative analyses of polyoxin D, a biological assay method using R. solani (known as Pellicularia sasaki S. Ito) as a test organism had been established,^7,8) but instrumental analytical methods such as highly specific HPLC and LC/MS/MS have been recently developed. The concentrations described in each study are based on quantitative values analyzed by one of those methods and to avoid confusion all concentrations are expressed in part per million (ppm).

The studies on polyoxins as an agricultural fungicide originated from a report showing the remarkable activity of two polyoxin components (A and B) against four plant pathogenic fungi, Alternaria kikuchiana S. Tanaka (A. kikuchiana), Cochliobolus miyabeanus Drechsler ex Dastur (C. miyabeanus), R. solani, and Pyricularia oryzae Cavara (P. oryzae), and the superior safety in mammals, fish, and plants.⁹⁾ Some of the polyoxins have no antifungal activity,¹⁰⁾ and others have different selectivity against filamentous fungi.¹¹⁾ However, polyoxin D broadly inhibits the mycelial growth of fungi, whether basidiomycetes, ascomycetes, or zygomycetes (Table 1). It is characterized by the highest activity against the genus Rhizoctonia at low concentrations of 1.562 ppm or less.¹¹⁾ On the other hand, there is a 10-fold difference in the EC₅₀ value (half maximal effective concentration) of polyoxin D against some field isolates of Botrytis cinerea Persoon (B. cinerea), which ranged between 0.59 ppm (minimum) and 5.8 ppm (maximum).¹²⁾ In addition, the EC₅₀ values against Colletotrichum species (C. nymphaeae Aa, C. fructicola Prihastuti, L. Cai & K. D. Hyde, C. siamense Prihastuti, L. Cai & K. D. Hyde, C. fioriniae R. G. Shivas & Y. P. Tan) isolated from peach (Prunus spp.) orchards vary depending on the strain, stage of fungal growth, and culture medium composition,¹³⁾ so further evaluation is necessary to determine the optimal activity. Polyoxin D is also active against apple tree diseases inducing wood decay, such as valsa canker caused by Valsa ceratosperma Maire (unpublished) and pink disease caused by Erythricium salmonicolor Burdsall.¹⁴⁾ The EC₅₀ value of polyoxin D against wood-rotting fungus (Sphaerobolus stellatus SS8 strain) is reported to be the lowest among the 26 fungicides tested.¹⁵⁾ The effect of polyoxin D on oomycetes such as downy mildew fungi and Phytophthora infestans (P. infestans) are inconsistent (unpublished) and will be discussed later.

The germ tubes of Alternaria mali Roberts (A. mali) are swollen in a conidial suspension treated with polyoxin D at more than 0.5 ppm,¹⁶⁾ and similar abnormal morphologies are observed in conidia and septum formation of Alternaria tomato Weber (A. tomato) treated with 10 ppm of a mixture of polyoxins D, E, and F.¹⁷⁾ In Fig. 1, a micrograph of the swollen germ tubes of A. mali conidia following treatment with 2 ppm of polyoxin D is shown, and such morphological changes are also observed in hyphae of C. miyabeanus at 100 ppm,¹⁸⁾ in germinating conidial of Alternaria brassicae (A. brassicae) at 50 ppm,¹⁹⁾ in conidial germ tubes of P. oryzae at 1.56–100 ppm,²⁰⁾ and in hyphae of P. oryzae at 0.1 ppm of polyoxin D.²¹⁾ The swollen germ tubes and hyphae take on a protoplast-like form¹⁸⁾; they swell to 2–3 times their size and become brittle.²²⁾ It is presumed that the cell walls become thin and are unable to withstand the osmotic pressure, resulting in rupture or lysis.¹⁸⁾ Similar morphological abnormalities caused by polyoxin D have also been observed in Candida species,^22,23) Saccharomyces cerevisiae X2180 (ATCC 26109)²⁴⁾ and S. cerevisiae SSY563-9B.²⁵⁾ However, the effective concentrations of this compound against the yeast-phase fungi are much higher than those against other fungi and are thus not practical for their control.

Fig. 1. Conidial germination of Alternaria mali treated with 2 ppm polyoxin D. Conidia provided from Apple Research Institute, Aomori Prefectural Industrial Technology Research Center, maintained in sterile water solution at 25°C in the dark after the treatment. ppm=part per million

It has been reported that the total conidial numbers of A. brassicae, including those with normal and abnormal morphologies, per colony are not appreciably decreased by polyoxin D treatments at 0.04–12.5 ppm.¹⁹⁾ However, the conidial numbers of A. tomato are reduced dose-dependently by a mixture of polyoxins D, E, and F at 10–30 ppm, and around 80% of the conidia show abnormal morphology at all doses.¹⁷⁾ Inhibition of sporulation of A. kikuchiana is also observed following treatments with polyoxins at concentrations between 0.125 and 1.0 ppm without affecting mycelial growth.¹⁶⁾ The conidial number of Fusarium fujikuroi Nirenberg treated with 15–150 ppm of polyoxin D is reduced dose-dependently without affecting mycelial growth.²⁶⁾ We have also confirmed that spraying 50 ppm of polyoxin D on cucumber leaves infected with powdery mildew (Podosphaera xanthii) inhibits conidial formation by 98.8% (Fig. 2), which is consistent with those reports.

Fig. 2. Inhibition of powdery mildew (Podosphaera xanthii) conidial formation on cucumber. Cucumber nursery inoculated with powdery mildew kept in a phytotron at 25/17°C (12 hr/12 hr) and 70% relative humidity. Seven days after inoculation, 50 ppm of polyoxin D zinc salt was applied as a spray solution and incubated for 4 days. Conidia from infected cucumber leaf that were adhered on slide glass viewed using microscopy (×100), and counted. ppm=part per million

Generally, fungal cell walls are composed of polysaccharides, and basidiomycetes and ascomycetes cell walls contain more than 10–30% chitin.²⁷⁾ Isono and Suzuki²⁸⁾ indicated the structural similarity between polyoxins and uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a precursor of chitin (Fig. 3). In addition, Isono et al.²⁹⁾ speculated that the L-dipeptide moiety of polyoxins forms a reversible hydrogen bond with chitin synthase. Later, Endo et al.¹⁸⁾ revealed that polyoxin D selectively reduces the amount of UDP-GlcNAc incorporated into the chitin fraction in an ascomycete Neurospora crassa Shear & B. O. Dodge, competitively inhibiting the chitin synthase. Polyoxin D inhibits the enzyme activity of chitin synthase in P. oryzae by more than 80% and leads to accumulation of UDP-GlcNAc in C. miyabeanus cells by 150–160% in comparison to the non-treated control. However, it has been demonstrated that polyoxin D does not affect the uptake of other cell wall components such as glucose, amino acids, and sodium acetate,²¹⁾ and the same was verified by a cell wall regeneration test in P. oryzae protoplasts (Table 2).²⁰⁾ The inhibition of chitin synthase activity by polyoxin D has also been confirmed in a zygomycete fungus Mucor rouxii Wehmer.³⁰⁾ Further, Ren et al.³¹⁾ demonstrate that polyoxin D occupies the binding site of UDP-GlcNAc in the chitin synthase (Chs2) of Candida albicans, and proposed another possibility that N-terminal amino acid of polyoxin D extends into the tunnel and inhibits the chitin formation by overlapping with the acceptor binding site or obstructing chitin transformation based on the binding structure analysis.

Fig. 3. Chemical structures of polyoxin D and UDP-GlcNAc

The GlcNAc uptake and the chitin synthesis inhibitory effects of polyoxin D varied in the two growth stages (spherules and mycelia) of the soil fungus Coccidioides immitis,³²⁾ and chitin synthase activity and membrane permeability changes during the fungal growth process have been suggested to affect the activity of polyoxin D. Further, it was discovered that the antifungal activity of polyoxin D against R. solani could be inhibited by dipeptides such as glycylglycine (MW: 132.12 g/mol).⁵⁾ Dipeptides at 500-fold higher concentrations than polyoxin A have been reported to inhibit polyoxin A uptake and inactivate its efficacy in A. kikuchiana, suggesting that dipeptides can competitively inhibit polyoxins since they are taken up by the cells through the same cell membrane permeation system as polyoxins.³³⁾

On the other hand, it has been revealed that iminoctadine tetraacetate exhibits synergistic effects with polyoxins by increasing the intracellular uptake of polyoxin B in polyoxin-resistant strains of Alternaria alternata Keissler (A. alternata), due to the ability of iminoctadine tetraacetate in inhibiting the cell membrane function and securing a new uptake pathway for polyoxin B.³⁴⁾ In a mixture of polyoxin D and iminoctadine albesilate, we obtained an interaction ratio of 2.30 calculated from the EC₅₀ value against B. cinerea on cucumber according to Gisi’s method,³⁵⁾ which is higher than the criterion of 1.5 indicating synergism (Table 3).

It was previously reported that polyoxin D inhibited the spread of lesions at a concentration of 10 ppm on cut apple (Malus pumila) branches inoculated with A. mali to induce Alternaria leaf spot disease,¹⁶⁾ and thereafter, many studies have been conducted on the treatment of leaves and fruits of crops, or entire fields. In rapeseed (Brassica campestris L.) inoculated with the black spot fungus A. brassicae, pre-treatment with 50 ppm of polyoxin D reduced both the number and area of lesions.¹⁹⁾ It was also observed that treatment of wheat (Triticum aestivum L.) leaves inoculated with the stem rust fungus, Puccinia graminis Persoon, using 100 ppm of polyoxin D reduced the colony area.³⁶⁾ In peach (Prunus persica) fruits inoculated with six different strains of Colletotrichum genus, the anthracnose fungi isolated from orchards in the US, treatment with polyoxin D at 100 ppm showed equal or superior efficacy as compared to penthiopyrad.¹³⁾ In a two-season field test on grape (Vitis vinifera L.) powdery mildew Erysiphe necator Schweinitz in a vineyard in India, two sprays of polyoxin D at three concentrations (10, 20, and 30 ppm) at 15-day intervals from first appearance of the disease improved the percent disease index of the lesions on leaves and fruits dose-dependently, and the yield at the standard dose (30 ppm) was higher than those of myclobutanil (MW: 288.78 g/mol, 20 ppm), flusilazole (MW: 315.39 g/mol, 20 ppm), and hexaconazole (MW: 314.21 g/mol, 50 ppm) by 20%, 17%, and 13%, respectively.³⁷⁾ A three-year field test on watermelon (Citrullus lanatus) in the US showed that polyoxin D (100 ppm) can be an alternative to chlorothalonil for powdery mildew and gummy stem blight control by spraying alone 7–10 times weekly.³⁸⁾ Further, it was demonstrated that polyoxin D treatment with 52 ppm was effective in controlling gummy stem blight in watermelon and melon (Cucumis spp.) seedling grown in greenhouse.³⁹⁾ Thus, polyoxin D is revealed to be comparable to conventional fungicides in controlling diseases of various crops when sprayed at a concentration range of 10–100 ppm. In general, crops defend themselves with papillae formation and secondary metabolite production once pathogens invade the cell to gain resistance against diseases caused by the pathogens. It would be necessary to verify that polyoxin D can suppress the growth of plant pathogens without inhibiting the plant’s own defenses.

The median lethal concentration (LC₅₀) of the polyoxin complex against the two-spotted spider mite (Tetranychus urticae) larvae was reported to be 14.2 ppm,⁴⁰⁾ and the LC₉₀ value against onion thrips (Thrips tabaci Lindeman) larvae was 8.6 ppm.⁴¹⁾ Although polyoxin complex is also registered as an insecticide in Japan, it has no activity on adult insects and egg hatching, and the effect on larvae is considered to be due to molting inhibition.⁴⁰⁾ However, there are no detailed research reports on its mode of action, and it is not listed in the chitin synthesis inhibitor group of the Insecticide Resistance Action Committee (IRAC) code classification. On the other hand, polyoxin D at 0.39 ppm inhibits insect chitin synthase, which is the similar target site in fungi.⁴²⁾ We have confirmed that polyoxin D has no utility as a spider mite control product (unpublished). Further, the LC₅₀ value against blowfly (Lucilia cuprina) larvae by digestion of polyoxin D is 10.4 ppm, which is about 35 times higher than the median inhibitory concentration of 0.3 ppm for GlcNAc uptake in the isolated integuments. It is speculated that absorption of polyoxin D in the larval midgut is very difficult, and even if it is absorbed, it is detoxified, so that its effect is unlikely to be detected in in vivo tests.⁴³⁾

Polyoxin D was confirmed to permeate the membrane of fungi, bind to chitin synthase, competitively inhibit the transport of the chitin precursor UDP-GlcNAc, make the germ tubes and hyphae of the fungi swollen, and show fungicidal activity in the previous chapter. However, it was reported that the swollen germ tubes of A. mali following exposure to polyoxins for more than 2 hr were able to grow normally again when cultured after removal of this compound.¹⁶⁾ In addition, it was demonstrated that the complete inhibitory dose of polyoxin D in A. brassicae was 3.12 ppm, which was 15 times higher than the significant inhibitory dose of 0.19 ppm,¹⁹⁾ and that while colony formation of Alternaria solani Sorauer (A. solani) and Sclerotium rolfsii Sacc. (S. rolfsii) was inhibited by polyoxin D, they still grew albeit slowly (Table 4).⁴⁴⁾ In liquid cultures with a daily dose of 20 ppm of polyoxins, Mitani and Inoue⁶⁾ observed that R. solani stopped growing for several days without lysis, and only the hyphal growth of P. oryzae was slowed. In light of this, they concluded that the action of polyoxins is fungistatic rather than fungicidal because both pathogens resumed growth after being transferred to fresh medium without polyoxins. In the germ tubes and hyphae of A. kikuchiana exposed to polyoxins, re-germination of the treated fungi from cells divided by new septa formed inside the inner layer of the swollen cell wall was observed microscopically,⁴⁵⁾ and the schematic diagram of morphological changes in filamentous fungi treated with polyoxin D is shown in Fig. 4.

Fig. 4. Schematic diagram of morphological changes in filamentous fungi swollen by polyoxin D

Although polyoxin D conveys crop protection through fungistatic activity and may vary depending on the infectious agent, it is still expected to reduce the density of spore-producing pathogens in the field by inhibiting conidiospore formation,^16,17,22) the spread of diseases due to secondary infection, as well as the risk of infection during the next crop season. Polyoxin D is considered to be beneficial in the integrated pest management system for both conventional and organic farmers.

Polyoxin D is water-soluble and can therefore be easily washed away by rain¹¹⁾; its inhibitory activity on pre-germinated conidia is greatly reduced by rainfall before drying after treatment. This is supported by the recovery of pathogenicity of A. mali¹⁶⁾ and the normal germination of A. brassicae conidia after treatment with polyoxins and subsequent washes with water.¹⁹⁾ On the other hand, the combination of polyoxin D with metal ions has been demonstrated to convey resistance to water.⁴⁶⁾ Therefore, polyoxin D is registered and marketed exclusively as polyoxin D zinc salt. Further, it was demonstrated that divalent metal ions promoted the intracellular uptake of polyoxin A by 2–3 times.³³⁾ Although Tewari & Skoropad¹⁹⁾ observed sufficient preventive effect of polyoxin D against rapeseed black spot disease caused by A. brassicae, poor rain resistance and short residual effect in field cultivation are still valid concerns, thus repeated applications may be necessary.

Another beneficial feature of polyoxins is the capability of deep penetration whereby treatment on one side of a rice leaf shows efficacy against R. solani infected on the opposite side.¹¹⁾ However, no significant systemic transfer from the roots or leaves in healthy plants is observed.¹¹⁾ Further, absorption and diffusion from wounds and cut ends of the plant body have been observed,^11,16) and there is a possibility of preventing post-harvest diseases in detached fruits or cut flowers.

There are few reports on the time-course activity of polyoxins after spraying, but polyoxins are relatively stable under ultraviolet light on rice plants and can maintain about 50–85% of their activity 6–9 days after application.¹¹⁾ In addition, it was reported that polyoxin complex showed a higher activity against tomato cercospora leaf mold induced by Pseudocercospora fuligena Deighton when sprayed at 7-day intervals than at 14-day intervals,⁴⁷⁾ and more than 60% of the preventive activity against tomato leaf mold induced by Passalora fulva U. Braun & Crous (P. fulva) was maintained for about 7 days.⁴⁸⁾ The half-life of polyoxin D residual activity on grape berries is reported to be 8 days (Fig. 5),³⁷⁾ and from those reports, the residual effect is considered to be about 7–14 days. On the other hand, it was reported that the addition of ester-based nonionic and cationic surfactants increased the adhesion of the polyoxin complex to the crops, which improved the treatment efficacy against cucumber powdery mildew (unidentified).⁴⁹⁾ A pinene-based adjuvant has also been shown to increase the effectiveness of polyoxin D against powdery mildew in cucumber (unpublished). Further research and development of polyoxin D are expected to be aimed at promoting adhesion to crops and uptake into the fungal cytoplasm.

Fig. 5. Dissipation of polyoxin D residue in/on grape berries³⁷⁾

The Fungicide Resistance Action Committee (FRAC)⁵⁰⁾ classifies polyoxins as cell wall biosynthesis inhibitors (FRAC code 19), which targets chitin synthase. No other fungicides are categorized in the same group. The risk of resistance is described as medium, and resistance management is required since polyoxin complex resistance has been reported in pear black spot caused by A. kikuchiana,⁵¹⁾ apple alternaria blotch caused by A. mali,⁵²⁾ and eggplant (Salanum melongena) leaf mold caused by Mycovellosiella nattrassii Deighton.⁵³⁾ A report on the A. kikuchiana strain that became resistant to polyoxin complex in Tottori Prefecture, Japan found that the rate of resistant strains in the Yonago area in 1971 reached 67.0% when 6 consecutive applications of polyoxin complex alone per season were made.⁵¹⁾ In a study of resistance genes, nine mutant Bipolaris maydis Shoemaker strains that induce corn (Zea mays) leaf spot and that had become resistant to polyoxin complex were isolated by chemical treatment.⁵⁴⁾ Two resistance genes (Pol2 and Pol5) were identified to be responsible for phenotypic alteration of the reddish brown pigmentation and polyoxin resistance.⁵⁵⁾ Resistance was determined to be due to gene mutations in the enzymes hydroxymethyl bilane synthase (HMBS) and ferrochelatase (FECH) involved in heme biosynthesis,⁵⁶⁾ but the mechanism of resistance is unclear.

Although polyoxin D resistance has not been confirmed yet, it may develop due to frequent exposure to this ingredient. The EC₅₀ values of A. solani and S. rolfsii sub-cultured in the lab for seven generations under exposure to polyoxin D was reported to be increased by more than 10 times.⁴⁴⁾ In a sensitivity study using 459 strains of B. cinerea isolated from 41 strawberry farms in the US where polyoxin D had never been used, 108 strains (23.5%) grew mycelia in media containing 5 ppm polyoxin D, and 29 strains (6.3%) were determined to have reduced sensitivity (RS) with a relative growth rate of 70% or more.¹²⁾ The RS strains were characterized by a slower growth rate and less spore formation than the sensitive strains, but they showed more sclerotinia formation, and these differences were also confirmed in the inoculated fruits.¹²⁾ Further, the EC₅₀ values of polyoxin D against 143 strains of A. alternata isolated from blueberry (Vaccinium spp.) fields in the US had a vast range (0.378–30.245 ppm), and a dose of 10 ppm significantly reduced the disease severity in fruits inoculated with those strains, but not the disease incidence (70.0–88.3%).⁵⁷⁾ It is anticipated that the properties of RS strains to polyoxin D will be clarified in the future, and it has been speculated that these strains harbor a pre-existing natural genetic resistance rather than developed an entirely new acquired resistance.³⁾

Incidentally, it has been demonstrated that polyoxins may also be effective against azoxystrobin- (quinone outside inhibitor) and triflumizole- (de-methylation inhibitor) resistant P. fulva that induce tomato (Solanum lycopersicum) leaf mold,⁴⁸⁾ as well as azoxystrobin- and thiophanate-methyl-(β-tubulin polymerization inhibitor) resistant Didymella bryoniae Rehm (D. bryoniae) which induce gummy stem blight.⁵⁸⁾ The authors have confirmed that there is no cross-resistance between polyoxin D and boscalid (succinate-dehydrogenase inhibitor) in P. fulva (Fig. 6).

Fig. 6. Efficacy of polyoxin D against SDHI-resistant Passalora fulva isolate. The isolate was provided from Gifu Prefectural Agricultural Technology Center. Three hours after spray treatment with each solution, the tomato plant was inoculated with a spore suspension (2×10⁵/mL) and kept at 23/17°C and 70% relative humidity in a phytotron followed by incubation at 23°C and 100% relative humidity in the dark for 48 hr. ppm=part per million

Biological control in crop protection has been recently developed as an alternative to chemical insecticides and acaricides with growing awareness of the environmental impacts on agriculture. Predatory mites have been put to practical use as biological control agents and are known to be damaged by exposure to some pesticides. Polyoxin complex is registered as an insecticide in Japan and reported to reduce the number of phytoseiid mite (Typhlodromips swirskii Athias-Henriot) which is one of the predatory mites.⁵⁹⁾ However, we found that the effect of polyoxin D on those mites was almost eliminated when it was sprayed to plants more than 3 days prior (unpublished). Although the influence of polyoxin D on the indigenous predatory mites is still unknown, since their main habitat in the orchard is in the undergrowth, it is unlikely that they will be exposed to high concentrations of the spray solution and the effect may be negligible.

It was shown that polyoxin D inhibited chitin synthesis in the midgut grinding material of the armyworm moth at low concentrations (0.52–1.04 ppm).⁶⁰⁾ However, polyoxin D had no effect in lepidopteran (Spodoptera litura) larvae by the oral administration at 150 ppm and in silkworm (Bombyx mori) by hypodermically injection.⁶¹⁾ In addition, no influence on the peritrophic membrane was found when polyoxin D was orally administered at 26 ppm to fly (Lucilia cuprina)⁶²⁾ or mosquito (Anopheles gambiae; Aedes aegypti)⁶³⁾ larvae, and no structural destruction of the midgut was observed in mosquito larvae when polyoxin D at 100 ppm was orally administered or injected into the thorax.⁶³⁾ Therefore, there is no evidence that polyoxin D has any effect on predatory mites and their natural preys.

Polyoxin complex has been reported to inhibit the mycelial growth of a fungus Metarhizium spp. that are parasitic to thrips larvae, however, it does not kill the parasitic fungus at practical concentrations.⁶⁴⁾ This effect is believed to be very minor or non-existent in practical application due to the different localization of the microbial pesticide and polyoxin complex treatment. The Metarhizium agents are generally sprayed at the base of the plant, and polyoxin complex is sprayed on stems and leaves, thus there would be very little interaction between the two agents. Although polyoxin complex showed fungal activity on isolated spores of the entomo-parasitic fungus Beauveria bassiana (B. brassiana), little effect of polyoxin complex formulations have been observed.⁶⁵⁾ On the other hand, Saha et al.⁶⁶⁾ has reported that polyoxin D at the concentrations of 30 and 60 ppm is compatible in vitro with almost biocontrol agents such as Trichoderma viride, Bacillussubtilis, Pseudomonas fluorescence, Ampelomyces quisqualis and Metarhizium anisopliae but incompatible with B. brassiana. Further studies using their formulated biocontrol agents would be required. In addition, the spore germ tube of the nematode-trapping fungus, Anthrobotrys oligospora, was swollen when treated with polyoxin D at 0.02–20 ppm, but the hyphae continued to elongate and its function was maintained.⁶⁷⁾ From those results, it is considered that polyoxin D may have some influence on predatory mites or microbial pesticides when they are used at the same time and should be used at an appropriate interval. In addition, since no effects on honeybees (Apis mellifera L) or flower-visiting insects such as marmalade hoverfly (Epistrophus balteatus), green lacewing (Chrysoperia carnea) and ladybird beetle (Coccinellidae) have been demonstrated,⁶⁸⁾ polyoxin D can be a suitable material for environmentally friendly agriculture.

Soil-borne pathogens in fields mainly infect the plant through roots, so the infection is hard to notice until foliar symptoms appear. Polyoxin D sprayed at a concentration of 22.2–44.4 ppm is effective in controlling cabbage head rot and lettuce bottom rot caused by R. solani as well as southern blight induced by S. rolfsii in onions (Allium cepa) and carrots (Daucus carota) (Table 5). Further, polyoxins were reported to show activity against the soil-borne pathogen Verticillium dahliae Klebahn at 10 ppm, but the fungal activity was reduced in a mutant lacking the protein kinase (VdPKC) gene which is essential for the expression of pathogenicity. This is a different reaction than observed for other fungicides.⁶⁹⁾ The half-lives of polyoxin D in aerobic non-sterile and sterilized soils are 15.9 and 59.2 days, respectively, which suggests that polyoxin D may be strongly adsorbed to soil particles (Table 6).⁶⁸⁾ It is possible that a certain amount of sprayed polyoxin D is transported to soil by rainfall and inhibits the soil-borne pathogens for about 2 weeks, but soil application or drench may be more effective.

Some major seed-borne diseases include leaf blight caused by Alternaria dauci J. W. Groves & Skolko, alternaria black spot caused by Alternaria radicina Meier, Drechsler & E. D. Eddy in carrots, alternaria sooty spot caused by A. brassicicola Wiltshire, black leg caused by Phoma lingam Desmazières in cabbage (Brassica oleracea L.), and verticillium wilt caused by V. dahliae in spinach (Spinacia oleracea). When there is a high infection rate of Alternaria sooty spot in cabbage seeds, a pesticide treatment on seed or sowing is recommended to prevent the spread of the disease.⁷⁰⁾ It has been reported that soaking alternaria sooty spot-infected cabbage seeds in a 2.5% polyoxin complex solution for 10 min is sufficient disease control that leads to a high germination rate.⁷¹⁾ Further, polyoxin complex spray treatment during the seedling stage of cabbage has been found to be effective against black leg.⁷²⁾ Polyoxin D treatment on seeds or seedlings planted in trays is anticipated to show a similar effect.

It has been reported that volatile components contained in the culture filtrate of Streptomyces cacaoi Waksman and Henrici ssp. cacaoi Waksman and Henrici M20 strain isolated from mangrove soil can inhibit the growth of Fusarium oxysporum Schlechtend,⁷³⁾ and that soil incorporation of Streptomyces spp. isolated from cabbage leaf surfaces can suppress Alternaria sooty spot caused by A. brassicicola.⁷⁴⁾ Bio-stimulants, which act directly on plants and improve their physiological conditions to improve immunity, relieve stress, and support healthy growth, have been globally used.⁷⁵⁾ The EU has clarified its definition and classification of bio-stimulants in by the European Fertilizer Regulation (EC) No. 2019/1009.⁷⁶⁾ A fermentation residue containing killed actinomycetes S. cacaoi var. asoensis results from the manufacturing process of polyoxin D. If the safety of the fermentation residue is confirmed in plant toxicity assessments, utilization of polyoxin D fermentation residue as a fertilizer or bio-stimulant is anticipated.

Decay caused by fungi including those in the genera Alternaria, Botrytis, Penicillium, Monilinia, Aspergillus, and Botryosphaeria has been generally observed in apples during storage and/or distribution in Japan.⁷⁷⁾ Polyoxin D is registered as a post-harvest fungicide in the US and the 5% SC formulation can be available for the prevention of post-harvest diseases in pome fruits, stone fruits, and pomegranates (Punica granatum) at concentrations of 13.1–59.4 ppm by in-line dip, drench, or aqueous spray treatment.⁷⁸⁾ According to many studies performed by Adaskaveg,⁷⁹⁾ although the effectiveness varied depending on the fruit variety, maturity, and pathogenic fungi, polyoxin D treatment on apple fruits 15–17 hr after inoculation showed high activity against Alternaria spp., and treatment within 10 hr after inoculation with B. cinerea was even more efficient. Further investigations on the optimal post-harvest treatment method and timing of polyoxin D for apples are expected.

Although polyoxin D zinc salt had not been permitted to remain in any crop in Japan, its acceptable daily intake was determined at 7.2 mg/kg body weight/day by Food Safety Commission of Japan in 2021,⁸⁰⁾ and thereafter, both maximum residue limit and pre-harvest interval in each crop would be defined based on the residual study data. We have observed that polyoxin D spray reduce not only damage caused by black spot disease on cherry (Prunus sp.) trees but also storage decay when the pathogen is experimentally inoculated in detached cherry fruits (Fig. 7). Some pre-harvest infected fruits may be collected with latent infection resulting in decay during distribution. Because it is difficult to prevent post-harvest decay with pre-harvest spray alone, a combination of pre- and post-harvest treatments is important for food waste reduction.

Fig. 7. Effect of polyoxin D on Alternaria leaf spot disease in pre- and post-harvest cherry fruit. Three cherry trees were sprayed with each fungicides for 3 times by approximately 10-day intervals before harvest. Six days after the final spray, 100 cherry fruits/tree in each treatment were investigated the lesions caused by Alternaria spp.. After the investigation, 50 cherry fruits/tree showing no lesions were detached and inoculated with 20 µL/fruit of spore suspension (4.2×10⁵/mL) of Alternaria spp.. These fruits were kept at 25°C and 100% relative humidity for 7 days and investigated the lesions. ppm=part per million

Aspergillus spp. and Fusarium spp. on grains such as corn produce mycotoxins and can cause serious illness to humans and livestock. Polyoxin D is reported to dose-dependently inhibit the production of aflatoxins from Aspergillus parasiticus Speare and fumonisins from Fusarium fujikuroi in the culture broth without the suppression of fungal growth. The observed fifty percent inhibitory concentrations in those fungi were 57 ppm and 21.9 ppm, respectively.²⁶⁾ However, treatment of wheat with 50 ppm of polyoxin D four times with 7–9 days interval from the flowering did not demonstrate any observable control of Fusarium blight or suppression of deoxynivalenol (unpublished), which is a major mycotoxin produced by Fusarium graminearum Schwabe or Fusarium culmorum Saccardo on wheat or corn. Further investigations are necessary to clarify the potential use of polyoxin D in mycotoxin control. The structure of fumonisin is very similar to AAL toxin which is produced by A. alternata and shows host-specific pathogenecity.⁸¹⁾ If polyoxin D could suppress the AAL toxin production in addition to the inhibition of chitin synthase, it would lead to the discovery of a new action site for polyoxin D.

After the discovery of polyoxins, chitin synthase has become one of the major targets in fungicide. Neo-polyoxin C was discovered and determined to be structurally identical to nikkomycin Z which is a chitin synthesis inhibitor.¹⁾ Two types of chitin synthases (Chs1 and Chs2) were detected in S. cerevisiae, and polyoxin D was found to have higher activity than nikkomycin Z against Chs2, which is required for septum formation, and in suppression of the number of viable cells.²⁵⁾ It has been reported that chitin synthases are classified into classes I to VII, and fungi have multiple chitin synthases with different functions.⁸²⁾ Polyoxin B is suggested to bind to Chs1, which has a repair function in S. cerevisiae at its nucleoside moiety, and to increase cell membrane permeability at the peptide moiety leading to inhibition of enzyme activity by more than 80%.⁸³⁾ Several studies have attempted to analyze the three-dimensional structure of a binding model of yeast chitin synthase and nikkomycin Z, and to clarify the mechanism of inhibition of chitin synthase.^83–85) It is anticipated that such approach will clarify the class of chitin synthase produced by fungi and their individual function, and the detailed action of polyoxins. On the other hand, the effect of polyoxins on fungi has been shown to be lower at the cellular level as compared to that at the enzyme level due to the poor uptake of polyoxins into the cell.⁸⁶⁾ Research has been conducted on the synthesis of derivatives to improve the uptake, and some hybrid chitin synthase inhibitors are found to show higher biological activity against certain pathogenic fungi.⁸⁷⁾

Oomycetes Pythium spp. and Phytophthora spp. cause difficult-to-control diseases that are very damaging even in greenhouse cultivation where crops are grown hydroponically.⁸⁸⁾ Although oomycetes were thought to carry cellulose instead of chitin in their cell wall, chitin and its synthesizing enzyme have been identified in P. infestans, and morphological changes such as swelling and bursts are observed when P. infestans are treated with nikkomycin Z.⁸⁹⁾ If these morphological alterations are due to inhibition of chitin synthase by nikkomycin Z, polyoxin D may also be able to inhibit P. infestans. If these pathogens can be controlled by polyoxin D treatment, this will lead to new applications in organic farming.

Polyoxin D has shown to suppress the expression of fum21, which encodes a fumonisin-producing protein that functions to stabilize cell wall traits, through the Slt2-MAPK (mitogen-activated protein kinase) pathway in F. fujikuroi.²⁶⁾ In addition, polyoxins are suggested to interfere with the cell wall integrity maintenance mechanism in V. dahliae by binding to VdPKC and inhibiting the signal transduction of osmotic stress.⁶⁹⁾ On the other hand, when the expression level of the gene, pkcA which encodes a MAPK that works downstream of PKC in the fungus A. nidulans, is suppressed, the penicillin synthesis and the cell integrity are reduced.⁹⁰⁾ Ichinomiya et al.⁹¹⁾ have observed that polyoxin B induces the growth delay in the pkcA-overproducing strain of A. nidulans, but not in wild type, and further studies are necessary to clarify the relationship between pkcA and polyoxin B. Polyoxin D may have multiple action sites other than chitin synthase in fungi. Further investigation is anticipated to clarify the involvement in the production of secondary metabolites such as mycotoxins by inhibiting the PKC-MAPK pathway.

Polyoxin D has antifungal activity against fungi and a unique mode of action by chitin synthase inhibition. The action is fungistatic and reduces the conidial production that represents the subsequent infectious source. Further, polyoxin D has the potential to control soil-borne and post-harvest diseases and to inhibit the mycotoxin production via a new mode of action. These novel findings are anticipated to bring forth new formulations or applications for agricultural purpose, although more than 60 years have passed since polyoxin D was discovered. In conclusion, polyoxin D is considered as an ideal agricultural fungicide that suitably addresses the environmental issues due to the least amount of harm to human, animals, plants, honeybees and flower-visiting insect.

We greatly appreciate Dr. Masaharu Kubota of the National Agriculture and Food Research Organization and Ms. Cynthia Smith of Conn & Smith, Inc. for their advice regarding the preparation of the manuscript. We are also deeply grateful to our colleagues in the Agrochemical and Animal Health Products Department, Kaken Pharmaceutical Co., Ltd. for the collection of literature and their meaningful consultation.

• 1) H. Osada: Discovery and applications of nucleoside antibiotics beyond polyoxin. J. Antibiot. (Tokyo) 72, 855–864 (2019).
• 2) Consideration of Eligibility for Registration of the New Pesticide Active Ingredient Polyoxin D Zinc Salt—DECISION MEMORANDUM—, US Environmental Agency Office. https://www3.epa.gov/pesticides/chem_search/reg_actions/registration/related_PC-230000_1-Jul-03.pdf (Accessed 7 June, 2024)
• 3) Polyoxin D Zinc Salt—Technical Evaluation Report, OMRI for the USDA national Organic Program, https://www.ams.usda.gov/sites/default/files/media/PolyoxinDZincSaltTR2022.pdf (Accessed 29 Jan., 2025)
• 4) L. G. Copping and J. J. Menn: Biopesticides: A review of their action, applications and efficacy. Pest Manag. Sci. 56, 651–676 (2000).
• 5) K. Isono: New aspect in research and development of agricultura antibiotics. Nippon Nogeikagaku Kaishi 63, 1351–1358 (1989) (in Japanese).
• 6) M. Mitani and Y. Inoue: Antagonists of antifungal substance polyoxin. J. Antibiot. (Tokyo) 21, 492–496 (1968).
• 7) H. Sakurai, T. Morita, H. Suzuki and K. Yoshida: Quantitative analysis of polyoxins with bioassay. Bull. Agric. Chem. Inspect. Stn. 9, 30–36 (1969) (in Japanese).
• 8) H. Sakurai, T. Shimada and H. S. Baba: Quantitative analysis of polyoxins with bioassay (supplemental report). Bull. Agric. Chem. Inspect. Stn. 10, 76–78 (1970) (in Japanese).
• 9) K. Isono, J. Nagatsu, Y. Kawashima and S. Suzuki: Studies on polyoxins, antifungal antibiotics part I. Isolation and characterization of polyoxins A and B. Agric. Biol. Chem. 29, 848–854 (1965).
• 10) K. Isono, J. Nagatsu, K. Kobinata, K. Sasaki and S. Suzuki: Studies on polyoxins, antifungal antibiotics part V. Isolation and characterization of polyoxins C, D, E, F, G, H and I. Agric. Biol. Chem. 31, 190–199 (1967).
• 11) S. Sasaki, N. Ohta, J. Eguchi, Y. Furukawa, T. Akashiba, T. Tsuchiyama and S. Suzuki: Studies on polyoxins, antifungal antibiotics. VIII. Mechanism of action on sheath blight of rice plant. Ann. Phytopathol. Soc. Jpn. 34, 272–279 (1968) (in Japanese).
• 12) M. E. Dowling, M.-J. Hu, L. T. Schmitz, J. R. Wilson and G. Schnabel: Characterization of Botrytis cinerea isolates from strawberry with reduced sensitivity to polyoxin D zinc salt. Plant Dis. 100, 2057–2061 (2016).
• 13) X. J. Hao, M. J. Hu, S. N. Chen and G. Schnabel: Challenges in assessing efficacy of polyoxin-D zinc salt against colletotrichum species. J. Plant Pathol. 99, 513–516 (2017).
• 14) Y. Saito and K. Hiroma: Occurrence of pink disease of apple tree and chemical control. Proceedings of Kanto-Tosan Plant Protection Society 34, 105–106 (1987) (in Japanese).
• 15) M. A. Fidanza and D. D. Davis: In vitro screening of fungicides to control artillery fungi. J. Environ. Hortic. 27, 155–158 (2009).
• 16) J. Eguchi, S. Sasaki, N. Ota, T. Akashiba, T. Tsuchiyama and S. Suzuki: Studies on polyoxins, antifungal antibiotics. IX. Mechanism of action on the diseases caused by Alternaria spp. Ann. Phytopathol. Soc. Jpn. 34, 280–288 (1968) (in Japanese).
• 17) N. Yoshioka, T. Kumagai and Y. Oda: Effect of polyoxin on the morphogenesis of the fungus Alternaria tomato, with the special reference photosporulation. Dev. Growth Differ. 17, 281–286 (1975).
• 18) A. Endo, K. Kakiki and T. Misato: Mechanism of action of the antifungal agent polyoxin D. J. Bacteriol. 104, 189–196 (1970).
• 19) J. P. Tewari and W. P. Skoropad: The effect of polyoxins B and D on Alternaria brassicae and the blackspot of rapeseed. Can. J. Plant Sci. 59, 1–6 (1979).
• 20) T. Teraoka, C. S. Vega, D. Hosokawa, M. Watanabe and K. Wakabayashi: Effect of polyoxin D on cell wall regeneration and biosynthesis in protoplasts from Pyricularia oryzae. J. Pestic. Sci. 13, 213–220 (1988).
• 21) N. Ohta, K. Kakiki and T. Misato: Studies on the mode of action of polyoxin D part II. Effect of polyoxin D on the synthesis of fungal cell wall chitin. Agric. Biol. Chem. 34, 1224–1234 (1970).
• 22) J. M. Becker, N. L. Covert, P. Shenbagamurthi, A. S. Steinfeld and F. Naider: Polyoxin D inhibits growth of zoopathogenic fungi. Antimicrob. Agents Chemother. 23, 926–929 (1983).
• 23) L. L. Hilenski, F. Naider and J. M. Becker: Polyoxin inhibits colloidal gold-wheat germ agglutinin labelling of chitin in dimorphic forms of Candida albicans. Microbiology (Reading) 132, 1441–1451 (1986).
• 24) B. Bowers, G. Levin and E. Cabib: Effect of polyoxin D on chitin synthesis and septum formation in Saccharomyces cerevisiae. J. Bacteriol. 119, 564–575 (1974).
• 25) E. Cabib: Differential inhibition of chitin synthetases 1 and 2 from Saccharomyces cerevisiae by polyoxin D and nikkomycins. Antimicrob. Agents Chemother. 35, 170–173 (1991).
• 26) T. Yoshinari, M. Watanabe and Y. Hara-Kudo: Cross-genus inhibitory activity of polyoxins against afratoxin production by Aspergillus parasiticus and fumonisin production by Fusarium fujikuroi. FEMS Microbiol. Lett. 369, fnac048 (2022).
• 27) T. Ichimiya and H. Horiuchi: Morphogenesis of filamentous fungi from the perspective of cell wall synthesis. Chemical and Biology 39, 255–264 (2001) (in Japanese).
• 28) K. Isono and S. Suzuki: The structures of polyoxins D, E, F, G, H, I, J, K and L. Agric. Biol. Chem. 32, 1193–1197 (1968).
• 29) K. Isono, S. Suzuki and T. Azuma: Semisynthetic polyoxins, aminoacyl derivatives of polyoxin C and their in vitro activity. Agric. Biol. Chem. 35, 1986–1989 (1971).
• 30) S. Barnichi-Garcia and E. Lippman: Inhibition of Mucor rouxii by polyoxin D: Effects on chitin synthetase and morphological development. J. Gen. Microbiol. 71, 301–309 (1972).
• 31) Z. Ren, A. Chhetri, Z. Guan, Y. Suo, K. Yokoyama and S. Y. Lee: Structural basis for inhibition and regulation of a chitin synthase from Candida albicans. Nat. Struct. Mol. Biol. 29, 653–664 (2022).
• 32) R. F. Hector and D. Pappagianis: Inhibition of chitin synthesis in the cell wall of coccidioides immitis by polyoxin D. J. Bacteriol. 154, 488–498 (1983).
• 33) M. Hori, K. Kakiki and T. Misato: Antagonistic effect of dipeptides on the uptake of polyoxin A by Alternaria kikuchiana. J. Pestic. Sci. 2, 139–149 (1977).
• 34) H. Sekido, T. Shimizu, I. Miura, S. Maeno, S. Hayashi and I. Nakayama: Synergistic mechanism of polyoxin B and iminoctadine triacetate in polybelin^® on polyoxin-resistant Alternaria alternata apple pathotype. J. Pestic. Sci. 21, 281–285 (1996).
• 35) U. Gisi, H. Binder and E. Rimbach: Synergistic interaction of fungicides with different modes of action. Trans. Br. Mycol. Soc. 85, 299–306 (1985).
• 36) W. K. Kim, R. Rohringer, D. J. Sambroski and N. K. Howes: Effects of blasticidin S, ethionine and polyoxin D on stem rust development and host-cell necrosis in wheat near-isogenic for gene Sr6. Can. J. Bot. 55, 568–573 (1977).
• 37) P. Deore, S. Hingmire, D. Shinde, A. Pudale, T. P. A. Shabeer, K. Banerjee, R. U. Thosar and S. Saha: Field bio-efficacy and residue dynamics of the fungicide polyoxin D zinc salt 5% SC in grape (Vitis vinifera). Int. J. Bio-Resour. Stress Manag. 12, 603–610 (2021).
• 38) J. G. Jones, R. C. Korir, T. L. Walter and K. L. Everts: Reducing chlorothalonil use in fungicide spray programs for powdery mildew, anthracnose, and gummy stem blight in melons. Plant Dis. 104, 3213–3220 (2020).
• 39) A. P. Keinath: Polyoxin D and other biopesticides reduce gummy stem blight but not antracnose on melon seedling. Plant Health Prog. 17, 177–181 (2016).
• 40) S. Kochi, T. Fujimaki, J. Tokumura and M. Hori: Biological activity of polyoxin against tetranychidae. 35th Annual Meeting of the Japanese Society Pesticide Science, p.93 (2010) (in Japanese).
• 41) S. Kochi, T. Fujimaki, T. Yano and J. Tokumura: Insecticidal activity of polyoxin against agricultural insect pest. 36th Annual Meeting of the Japanese Society Pesticide Science, p.116 (2011) (in Japanese).
• 42) B. A. Sowa and E. P. Marks: An in vitro system for the quantitative measurement of chitin synthesis in the cockroach: inhibition by TH6040 and polyoxin D. Insect Biochem. 5, 855–859 (1975).
• 43) I. F. Turnbull and A. J. Howells: Effects of several larvicidal compounds on chitin biosynthesis by isolaetd larval integuments of the sheep blowfly Lucilia cuprina. Aust. J. Biol. Sci. 35, 491–503 (1982).
• 44) R. Maria and S. B. Sullia: Acquired resistance of Alternaria solani and Sclerotium rolfsii to Polyoxin-D. J. Phytopathol. 116, 60–66 (1986).
• 45) H. Ishizaki, K. Mitsuoka, M. Kohno and H. Kunoh: Effect of polyoxin on fungi III, electron microscopic observation of myceria of Alternaria kikuchiana Tanaka. Ann. Phytopathol. Soc. Jpn. 42, 35–41 (1976).
• 46) S. Suzuki, K. Hashimoto and K. Murata: Agricultural fungicide. Japanese Patent Application, 793925 (1975) (in Japanese).
• 47) H. Taguchi, H. Suzuki and K. Kuroda: Efficacy of fungicides in the control of Pseudocercospora fuligena on Tomato. Kansai Byochugai Kenkyukaiho 54, 7–12 (2012) (in Japanese).
• 48) H. Watanabe and H. Horinouchi: Preventive effect of several fungicides to tomato leaf mold caused by Passalora fulva. Kansai Byochugai Kenkyukaiho 53, 63–65 (2011) (in Japanese).
• 49) N. Orihara, I. Uekusa and K. Kusano: Differential effect of several spreaders added to fungicides on powdery mildew of cucurbits. Annual Report of the Kanto-Tosan Plant Protection Society 46, 43–45 (1999) (in Japanese).
• 50) FRAC Code List© 2024: Fungal control agents sorted by cross-resistance pattern and mode of action (including coding for FRAC Groups on product labels), https://www.frac.info/docs/default-source/publications/frac-code-list/frac-code-list-2024.pdf (Accessed 29 Jan., 2025)
• 51) H. Udagawa: Occurrence of polyoxin-resistant black spot fungus of Japanese pear and countermeasures. Plant Protection 29, 189–193 (1975) (in Japanese).
• 52) Y. Onuma, T. Sanada and J. Eguchi: Polyoxin resistant strain of alternaria leaf spot in apple. Annual Report of the Society of Plant Protection of North Japan 24, 70 (1973) (in Japanese).
• 53) H. Ikeda, S. Tanaka, Y. Kajitani and T. Nakamura: Studies on fungicide resistance of vegetable diseases (3) Occurrence of polyoxin resistant strains of Mycovellosiella nattrassii deighton, mycovellosiella leaf mold of eggplant, and their chemical control. Bulletin of Fukuoka Agricultural Research Center, Series B (Horticulture) No.9, 7–12 (1989) (in Japanese).
• 54) A. Gafur, C. Tanaka, K. Shimizu, S. Ouchi and M. Tsuda: Genetic analysis of Cochliobolus heterostrophus polyoxin-resistant mutants. Mycoscience 39, 155–159 (1998).
• 55) C. Tanaka, K. Shimizu, A. Gafur and M. Tsuda: Polyoxin resistance of reddish-brown laboratory mutants of Cochliobolus heterostrophus. J. Gen. Plant Pathol. 68, 141–146 (2002).
• 56) D. Chen, H. Matsumoto, Y. Kitade, K. Izumitsu and C. Tanaka: Genetic analyses of reddish-brown polyoxin-resistant mutants of Bipolaris maydis. Mycoscience 59, 236–246 (2018).
• 57) F. Wang, S. Saito, T. J. Michailides and C. L. Xiao: Fungicide registance in alternaria alternata from blueberry in California and its impact on control of alternaria rot. Plant Dis. 106, 1446–1453 (2022).
• 58) T. Miyamoto, Y. Fujimoto, Y. Tomita and T. Ogawara: Distribution of Podosphaera xanthii, Didymella bryoniae and Pseudoperonospora cubensis isolates resistant to several fungicides on melon in Ibaraki prefecture. Research Report from the Horticultural Research Institute of the Ibaraki Prefectural Agricultural Center, 23, 17–25 (2017) (in Japanese).
• 59) M. Miyata: Affect of insecticide for predacious mite Amblyseius sawirskii. Plant Protection 64, 45–49 (2010) (in Japanese).
• 60) M. Tada, Y. Matsumoto, T. Mitsui, C. Nobusawa and J. Fukami: Inhibition of chitin synthesis by 1-(3,5-dichloro-2,4-difluorophenyl)-3-(2,6-difluorobenzoyl)urea (CME-134) in the cabbage armyworm, Mamestra brassicae L. J. Pestic. Sci. 11, 189–195 (1986).
• 61) T. Arakawa, F. Yukuhiro and H. Noda: Insecticidal effect of a fungicide polyoxin B on the larvae of Bombyx mori (Lepidoptera: Bombycidae), Mamestra brassicae, Mythimna separata, and Spodoptera litura (Lepidoptera: Noctuidae). Appl. Entomol. Zool. 43, 173–181 (2008).
• 62) R. L. Tellam and C. Eisemann: Chitin is only a minor component of the peritrophic matrix from larva of Lucilia cuprina. Insect Biochem. Mol. Biol. 30, 1189–1201 (2000).
• 63) M. J. Edwards and M. Jacobs-Lorena: Permeability and disruption of the peritrophic matrix and caecal membrane from Aedes aegypti and Anopheles gambiae msquito larvae. J. Insect Physiol. 46, 1313–1320 (2000).
• 64) K. Shirotsuka: Effect of chemical pesticides on an entomopathogenic microbial pesticide, Metarhizium anisopliae. Plant Protection 72, 26–30 (2018) (in Japanese).
• 65) M. Kubota, Y. Iida and S. Yamanaka: Annual Report of the Kanto-Tosan Plant Protection Society 67, 90 (2020) (in Japanese).
• 66) S. Saha, I. S. Sawant, S. B. Pawar, D. S. Yadav and Y. Ranade: Compatibility of polyoxin D zinc salt 5% SC against various biocontrol agents used in grapes. J. Mycopathol. Res. 58, 33–38 (2020).
• 67) Y. Persson, B. Nordbring-Hertz and I. Chet: Effect of polyoxin D on morphogenesis of the nematode-trapping fungus Anthrobotrys oligospora. Mycol. Res. 94, 196–200 (1990).
• 68) Polyoxin D Zinc Salt, Proposed Registration Decision, PRD2017-03, https://www.canada.ca/content/dam/hc-sc/migration/hc-sc/cps-spc/alt_formats/pdf/pest/part/consultations/prd2017-03/PRD2017-03-20eng.pdf (Accessed 29 Jan., 2025)
• 69) D. Wang, Z. Zhao, Y. Long and R. Fan: Protein kinase C is involved in vegetative development, stress response and pathogenicity in Verticillium dahlia. Int. J. Mol. Sci. 24, 14266 (2023).
• 70) M. Kubota: Ecology and control of sooty mold in cabbage plug seedlings. Plant Protection 64, 786–789 (2010) (in Japanese).
• 71) K. Kuroda and A. Tomikawa: Occurrence and control of alternaria sooty spot on plug seedlings of cabbage. Kansai Byochugai Kenkyukaiho 40, 121–122 (1998).
• 72) T. Nakano: Infection routes and control of cabage black leg in plug nursery. Bull Nara Agr. Exp. Sta 34, 37–42 (2003) (in Japanese).
• 73) T. Janaki: Biocontrol of Fusarium oxysporum in Unsterilized Soil by Novel Streptomyces cacaoi subsp cacaoi [M20]. Int. J. Pharm. Pharm. Sci. 9, 78–83 (2017).
• 74) N. Hassan, S. Nakasuji, M. M. Elsharkawy, H. A. Naznin, M. Kubota, H. Ketta and M. Shimizu: Biocontrol potential of an endophitic Streptomyces sp. strain MBCN152-1 against Alternaria brassicicola on cabage plug seedlings. Microbes Environ. 32, 133–141 (2017).
• 75) M. Narusaka and Y. Narusaka: How can biostimulants make a contribution to plant protection? Nihon Noyaku Gakkai Shi 47, 69–72 (2022) (in Japanese).
• 76) Regulation (EU) 2019/1009 of the European Parliament and of the Council of 5 June 2019 laying down rules on the making available on the market of EU fertilising products and amending Regulations (EC) No 1069/2009 and (EC) No 1107/2009 and repealing Regulation (EC) No 2003/2003 (Text with EEA relevance), http://data.europa.eu/eli/reg/2019/1009/oj (Accessed 29 Jan., 2025)
• 77) M. Tsuda: Fungi involved in postharvest deterioration of fruits and vegetables. Chemical and Biology 35, 656–661 (1997) (in Japanese).
• 78) U. S. EPA: Polyoxin D Zinc Salt 5SC Post-Harvest Fungicide, Registration Number 68173-5, Dec. 11 (2014).
• 79) J. E. Adaskaveg: Evaluation of New Bactericides for Control of Fire Blight of Apples caused by Erwinia amylovora and Evaluation of New Postharvest Fungicides for Pome Fruits “Annual Report 2013–2014,” California Apple Commission, pp.16–25 (2013).
• 80) Evaluation Report of Polyoxin D Zinc Salt Ver.2, Food Safety Commission of Japan, (2023) (in Japanese). https://www.mhlw.go.jp/content/11120000/001166063.pdf (Accessed 29 Jan., 2025)
• 81) Y. Akagi, T. Tsuge and M. Kodama: Evolution of pathogenicity in the alternaria plant pathogens. Plant Protection 67, 147–151 (2013) (in Japanese).
• 82) R. Liu, C. Xu, Q. Zhang, S. Wang and W. Fang: Evolution of the chitin synthase gene family correlates with fungal morphogenesis and adaption to ecological niches. Sci. Rep. 7, 44527 (2017).
• 83) D. D. Chen, Z. Wang, L. Wang, P. Zhao, C. Yun and L. Bai: Structure, catalysis, chitin transport, and selective inhibition of chitin synthase. Nat. Commun. 14, 4776 (2023).
• 84) Y. Ye, Z. Guan, X. Zhu and G. Yang: Chitin synthase: A potential agricultural and therapeutic target. Advanced Agrochem 1, 87–88 (2022).
• 85) Y. Wu, M. Zhang, Y. Yang, X. Ding, P. Yang, K. Huang, X. Hu, M. Zhang, X. Liu and H. Yu: Structures and mechanism of chitin synthase and its inhibition by antifungal drug nikkomycin Z. Cell Discov. 8, 129 (2022).
• 86) M. Winn, R. J. Goss, K. Kimura and T. D. Bugg: Antimicrobial nucleoside antibiotics targeting cell wall assembly: Recent advances in structure–function studies and nucleoside biosynthesis. Nat. Prod. Rep. 27, 279–304 (2010).
• 87) L. Zhai, S. Lin, D. Qu, X. Hong, L. Bai, W. Chen and Z. Deng: Engineering of an industrial polyoxin producer for the rational production of hybrid peptidyl nucleoside antibiotics. Metab. Eng. 14, 388–393 (2012).
• 88) S. Kusakari: Etiology and protection of pythium root rot disease in hydroponics. Plant Protection 65, 82–87 (2011) (in Japanese).
• 89) S. Klinter, V. Bulone and L. Arvestad: Diversity and evolution of chitin synthases in oomycetes (Straminipila: Oomycota). Mol. Phylogenet. Evol. 139, 106558 (2019).
• 90) M. Herrmann, P. Spröte and A. A. Brakhage: Protein Kinase C (PkcA) of Aspergillus nidulans is involved in Penicillin production. Appl. Environ. Microbiol. 72, 2957–2970 (2006).
• 91) M. Ichinomiya, H. Uchida, Y. Koshi, A. Ohta and H. Horiuchi: A protein kinase C-encoding gene, pkcA, is essential to the viability of the filamentous fungus Aspergillus nidulans. Biosci. Biotechnol. Biochem. 71, 2787–2799 (2007).