How does kasugamycin-copper or -mancozeb mixtures compare to copper-mancozeb?

Kasugamycin (tradename Kasumin) was registered in 2018 for managing walnut blight and bacterial canker and blast on sweet cherry. The bactericide was already federally registered for fire blight on pome fruit, but in 2018, registration for this disease was also approved in California. Kasugamycin is a unique bactericide because it is not used in animal or human medicine. Environmental monitoring studies have shown that it does not select for human bacterial pathogen resistance with uses in plant agriculture. Furthermore, kasugamycin has its own Fungicide Resistance Action Committee (FRAC) Code 24 or mode of action that is different from other registered plant agricultural bactericides like streptomycin (FRAC Code 25) and oxytetracycline (FRAC Code 41). Kasugamycin meets new toxicology standards for pollinating insects (e.g., honey bees), it has a low animal toxicity with a “Caution” rating and a 12 h re-entry time on the label. As with any cautionary pesticide, mixers and applicators need to have standard personal protective equipment (PPE) when handling the bactericide.

Copper is classified as FRAC Code M1 for the first element historically used for fungal and bacterial disease control. Copper affects many physiological pathways in plant pathogens and is classified as having a multi-site (M) mode of action. Not many bactericides have been developed for managing plant bacterial diseases, and fewer have been registered. Thus, there has been a great dependency on copper. Because of the multi-site classification, many agriculturalists thought that plant pathogens would not develop resistance to copper. Unfortunately, after many years of usage, bacterial pathogens such as the walnut blight pathogen, Xanthomonas arboricola pv. juglandis (Xaj), have developed resistance to copper. This is a direct result of overuse of one active ingredient (i.e., copper) and being limited with the lack of bactericides available to apply modern approaches to resistance management such as rotating between active ingredients with different modes of action and limiting the total number of applications of any one mode of action per season as part of following “RULES” (http://ipm.ucanr.edu/PDF/PMG/fungicideefficacytiming.pdf). Over-usage of any one active ingredient, such as copper, can create other environmental issues including soil contamination, orchard water-runoff, higher concentrations in watersheds, and potential crop and non-crop phytotoxicity especially in perennial crop systems.

After the industry used copper exclusively for approximately 50 years (1930s to 1980s), copper-maneb (e.g., Manex) mixtures were first identified for use on walnut in 1992 and emergency registrations ensued for 22 years before a full registration was obtained for the related compound mancozeb in 2014. The walnut industry and University of California (UC) researchers knew that more alternatives were needed, otherwise someday the pathogen would develop resistance to copper-mancozeb. Because copper resistance had already developed, this selection pressure is maintained and resistance levels are increasing even when mancozeb is used in the mixture, because copper has been the only tank mix option. In effect, resistance management is not being effectively practiced since copper-resistance already exists and the use of mancozeb (M3) is selecting for resistant strains of the bacterial pathogen to the mancozeb mode of action. Having only one treatment (i.e., mancozeb) available to manage a disease not only can limit crop production each season but could economically devastate the entire industry by making harvests sporadic and inconsistent, lowering crop quality, and preventing profitability. Growers and the entire walnut industry consider walnut blight a threat to the industry and their livelihood.

Why do we need kasugamycin for managing walnut blight?

There is a great need to develop other modes of action for managing bacterial diseases including walnut blight that can be integrated into management programs. Kasugamycin was identified, developed, and registered for the purpose of resistance management, reducing over-usage of any one mode of action, and sustaining the walnut industry of California. The aminoglycoside bactericide has a unique mode of action (FRAC Code 24) as stated above and can be used in combination with copper or mancozeb. When kasugamycin is used in combination with mancozeb, resistance management is being practiced since resistance has not been found in Xaj pathogen populations to either mode of action.

Use on Walnuts

Kasugamycin is labeled as Kasumin for managing walnut blight at 64 fl oz/A in a minimum of 100 gal water/A for ground application. The full 64 fl oz per acre labeled rate for kasugamycin should always be used. Adjuvants that are stickers may also be used, whereas spreaders and penetrants should be avoided. Reduced spray volumes may be utilized for small trees provided that the volume of water is sufficient to provide good coverage of treated foliage. Applications should be initiated when conditions favor disease development. This is the same timing as for copper-mancozeb. In orchards with a history of the disease and when high rainfall is forecasted, applications should be initiated at 20-40 percent catkin expansion. Under less favorable conditions for disease (i.e., low rainfall forecasts and minimal dews), applications should start at 20-40 percent pistillate flower expansion (also known as the “prayer stage”). The preharvest interval is 100 days or approximately mid- to late June depending on the walnut cultivar harvest date. The minimal re-application interval is seven days. The current labeled uses of Kasumin allows for two applications or 128 fl oz of product per season with a label change for up to four (256 fl oz) per season planned later this year. Still, only two consecutive applications will be allowed without rotating to other modes of action. Alternate row applications, applications in orchards that are being fertilized with animal waste/manure, or animal grazing in orchards treated with Kasumin are not allowed. The first restriction is to prevent selection of resistant isolates of the target pathogen, Xaj; whereas, the latter two restrictions are to ensure that the selection of non-target, human-pathogen bacteria is prevented.

For walnut blight management, the best way to use the bactericide is in combination with mancozeb or copper. Application management strategies for a four- or five-spray mixture, rotation program include, but are not limited to, the following:

A) Copper/mancozeb—kasugamycin/mancozeb—kasugamycin/copper—copper/mancozeb

B)  Copper/mancozeb—kasugamycin/mancozeb—copper/mancozeb—kasugamycin/copper — copper/mancozeb

How do kasugamycin treatments compare to copper-mancozeb treatments in managing disease?

The research used to develop kasugamycin was based on a 7- to 10-day re-application interval. The reason for this was that Kasumin is locally systemic or translaminar and thus, is less likely to be re-distributed. With new growth increasing the canopy volume weekly in the spring as walnut trees come out of dormancy, multiple and frequent applications are necessary. Kasugamycin-mancozeb mixtures applied in our research trials were often the most effective of all treatments evaluated.

In general, bactericides have a short residual life of a few days to a week or two. In toxicology in-vitro testing, Xaj is only moderately sensitive to kasugamycin with a mid-range to high minimum inhibitory concentration (MIC) value. When kasugamycin is mixed with mancozeb, the MIC of the mixture is approximately 5 parts per million (ppm). Kasugamycin is applied at 64 fl oz per 100 gal or 100 ppm. Thus, the labeled rate of kasugamycin-mancozeb mixtures are approximately 20X of the MIC value for Xaj. Because of the short residual activity and a moderate buffering residue (20X), the rotation of bactericide mixtures containing kasugamycin described above need to be applied in 7- to 10-day intervals.

Kasugamycin and Resistance.

Resistance is a relative term indicating a change in sensitivity to an inhibitory compound. A moderately high MIC for a bactericide does not mean that the pathogen is resistant. We have conducted baseline studies with kasugamycin, kasugamycin-copper, and kasugamycin-mancozeb for Xaj with MIC values of 20, 8.3, 5.3 ppm, respectively. This was done before the bactericide was registered in California to determine any change in sensitivity after registration and commercial usage. To date, resistance has not been found and isolates evaluated are all within the baseline distributions.  Still, with a single site mode of action compound such as kasugamycin, there is a risk for selecting resistant sub-populations of the pathogen especially when resistance management strategies are not employed. This is the reason why we developed the mixture-rotation programs suggested above.

Conclusions

The integration of bactericides with different modes of action and application strategies of rotations of mixtures of bactericides with different modes of action with forecasting tools such as XanthoCast (http://www.agtelemetry.com/) should provide the stewardship necessary for having the tools available for managing walnut blight for years to come. The hope with the Kasumin registration is to provide resistance management and prevent or reduce the risk of resistance to copper-mancozeb while new approaches can be developed and integrated to protect both of these compounds. Walnut blight is the most serious disease impacting growers in California and multiple tools like kasugamycin, copper, and mancozeb need to be available to maintain a successful industry.

White rot is caused by the fungus, Sclerotium cepivorum, which survives for decades in the soil as poppy seed-sized resting structures. If soil temperatures are between 50o and 75oF, compounds produced by onions and garlic trigger the fungus to break dormancy. Infection results in a soft rot of the garlic head or onion bulb, which will produce a white fluffy growth and then the poppy seed-like resting structures. Very few resting structures (only two in about a pint of soil) can result in losses in these crops. Thousands of resting structures may be produced on each diseased plant. Therefore, the levels of this pathogen in the soil can increase very rapidly in fields with a susceptible crop, which is limited to onions, garlic and a few relatives. The resting structures are spread within a field with tillage equipment and are moved to other fields with anything that moves soil. It can also be moved into new areas on garlic planting material.

For many years, the primary approach to white rot management was avoidance of infested fields. However, there are now more than 21,000 acres known to be infested with this pathogen and it is in areas where garlic and onions are important crops so it would limit production of these crops to completely avoid infested fields.

Sanitation

Sanitation is an important approach to limit spread. Cleaning equipment between fields will not only reduce risk of movement of the white rot pathogen through a production area but also limit risk of other soilborne diseases. Planting white rot-free garlic planting material is critical in keeping the pathogen out of fields that are not infested.

Disease Management
Several approaches to managing this disease hold promise. Metam applications can reduce soil inoculum levels but soil preparation and moisture conditions are critical in optimizing efficacy. Use of materials that emit compounds like those produced in the roots of onions and garlic to trigger germination of the resting structures in the absence of a host and starve out holds promise. However, additional work is needed to refine this approach for more reliable results than what has been observed experimentally. Research efforts now are focused on quantification and increasing concentrations of active volatile compounds in onion and garlic containing materials, identifying the levels needed to trigger germination and the specifics of effective approaches in applying these materials in the field.
Some fungicides applied in the trench where the planting material is dropped has consistently reduced incidence and severity of white rot. Fungicides applied through drip irrigation systems were not effective. Three years of studies were conducted in which fungicides were applied through drip irrigation systems with tubing either on the surface shallowly buried or buried at six inches and the treated plots were always the same as the untreated control.

Commercial Field Evaluation
During the 2016-17 and 2017-18 production seasons, fungicides were evaluated in a commercial field naturally infested with white rot in Fresno County. Fungicides in three conventional cate

gories were tested and in 2017-18, a non-living fermentation product with reported systemic acquired resistance activity was also included (Table 1, see page 13.) On 20 November 2016 and 11 November 2017, California late garlic was hand transplanted following the treatment of a 6-inch band of the trench into which the garlic cloves were dropped. The plots were rated on a scale from 0 to 10 with 0 being symptomless and 10 being collapsed. At maturity, 17 feet of each plot were hand dug and weighed, and per acre production was calculated. Data was subjected to Analysis of Variance and means were separated with Least Significant Difference P=0.05.

Under the conditions of these studies, most treatments had lower levels of above-ground symptoms than the untreated control. In the 2016-17 study, disease was severe and there were also significant differences in yields among treatments (Table 2, see page 14). The treatment with the highest yield was 95 percent higher (more than four tons per acre) than the untreated control. Disease pressure was lower in 2017-18 and there were no yield differences among treatments (Table 3). Tebuzol, Fontelis, A19649 and Rhyme 14 fluid oz/a consistently had lower levels of disease than the untreated control. Cannonball with SP2700 and Velum One demonstrated efficacy in the season that these materials were included in the test.

Fungicides are not intended to be the only approach to management of this disease and are not likely to provide commercially acceptable levels of control as the soil inoculum levels continue to increase. In addition, risk of the pathogen becoming resistant to the fungicides increases with increased and repeated use. Fungicides in three different groups have now consistently demonstrated efficacy and trials are underway evaluating different approaches to management of this production issue.

The research mentioned in this article was supported by the California Garlic and Onion Research Advisory Board and by industry donors.

Introduction: Apple Production, Storage and Rots
Apples have an estimated annual farm gate value of nearly $4 billion dollars in the United States, with downstream revenues exceeding $15 billion (US Apple Association). Apples are stored for extended periods of time (up to six months at 1°C in air, and for one year maximum in controlled atmosphere) to preserve their quality and provide fruit yearlong to meet customer demands. During storage, fungal rots can cause significant amounts of decay resulting in product losses, reduced quality, and lower economic returns for producers. The three most problematic rot causing fungi in the United States are Botrytis cinerea (gray mold), Penicillium spp. (blue mold), and Colletotrichum spp. (bitter rot) (Figures 1A-C). Both gray mold and bitter rot occur in the field and during storage. However, Penicillium expansum and other Penicillium spp., are found exclusively in storage and are also economically important (Xiao and Boal, 2009). The focus of this article will be on the blue mold fungus, but the information contained here is applicable to other fungal rot pathogens as well.

Blue Mold Biology
A survey of postharvest diseases in Washington State revealed that blue mold accounted for 28 percent of decay in storage (Kim and Xiao, 2008). Blue mold is characterized by a soft, watery rot that is light brown in color accompanied by the appearance of blue-green colored conidia on the fruit surface that develops at advanced stages of decay. P. expansum and other Penicillium spp. do not directly infect fruits, as they require wounds often caused by stem punctures and severe bruises that occur before, during, and after harvest (Figure 1A). Blue mold spreads by spores that are produced terminally in chains on the surface of whorled conidiophores (Figures 2A and B, see page 6). Stem punctures, during harvest and handling, provide places for rot to occur, but the fungus can also enter natural openings like lenticels, open calyx/sinus, and stem pull areas. Penicillium spp. also produce mycotoxins, such as patulin, citrinin, and penicillic acid, that pose potential human health risks when blue mold infected fruit are used to make juices and other processed products (Figures 3A-C). However, of the three mycotoxins, only patulin is regulated by the Food and Drug Administration (FDA) and European Food Safety Authority (EFSA) with a maximal allowable limit of 50 parts per billion (50 µg/kg-1) and 10 µg/kg-1 for babies and young infant products (European Union (EU), 2006).

Decay Management

Postharvest fungicides

Long term storage, coupled with lack of host resistance in commercial apple fruit cultivars, provides limited options but to rely on fungicides to manage postharvest decay of apple fruit (Rosenberger, 2012). Application of postharvest fungicides depends on the stage of product handling, and is typically made by bin drenching before storage, sorting line sprays, dips in flume water, together with fruit waxing, or by thermofogging storage rooms (Figures 4A-C, see page 9). There are four postharvest fungicides (Academy, Mertect, Penbotec, Scholar) registered and being used in the United States for apple fruits to manage postharvest decay. Both Scholar (active ingredient, fludioxonil) and Penbotec (active ingredient, pyrimethanil) were labeled for postharvest use in 2004 (Xiao and Boal, 2009). Recent reports indicate reduced efficacy of these materials that have resulted in increased blue and gray mold decay in commercially stored apple fruit in Pennsylvania and Washington State (Amiri et al., 2017, Yan et al., 2014; Gaskins et al., 2015). Thiabendazole (TBZ active ingredient, Mertect®), was labeled for blue mold management in 1968 and is applied primarily as a drench. Consequently, repeated long term use of TBZ has resulted in resistant Penicillium spp. for multiple apple growing regions of the United States (e.g. New York, Pennsylvania, Maryland) and in British Columbia (Rosenberger et al., 1990; Sholberg and Haag, 1996; Jurick II personal observations). A new product (under the name Academy) was introduced in 2016, containing two single site mode of action fungicides, fludioxonil & difenoconazole, to manage postharvest decay on apples.

Sanitation

Research studies have shown that fruit bins harbor fungal spores that serve as a source of inoculum to cause rot. Implementing physical (steam) or chemical (peroxyacetic acid, quaternary amines, etc.) sanitation methods in the packinghouse to reduce inoculum levels, reduces the incidence of fruit decay. While not commonly a stand-alone tool for postharvest rot management, bin sanitation complements existing chemical controls and helps to ensure their efficacy as resistant populations are kept in check (Rosenberger, 2012; Sholberg 2004; Hansen et al., 2010). Most inoculum of Penicillium spp. comes from the orchard soil and leaf litter as this pathogen can survive very effectively as a saprophyte (Lennox et al., 2003). Therefore, bins contaminated with soil and litter introduce this fungus into the packinghouse environment which is the source for the blue mold epidemic (Figure 5A-C, see page 10). Treating bins with steam, cleaning packinghouse walls and floors with quaternary amines or peroxyacetic acids, and maintaining proper chlorine levels in sizing flumes are all important measures that can have a positive impact in managing decay (Lennox et al., 2003). Hence, studies involving quantification of blue mold spores on bin surfaces in the Pacific Northwest region concluded that bin sanitation should be a component in an integrated decay management plan (Sanderson, 2000).

Fungicide resistance monitoring

Monitoring fungicide resistance in rot fungi is critical to maintain the efficacy of single-site mode of action fungicides. This is based on developing both, conventional and molecular-based methods, to detect fungicide-resistant pathogen populations. Routine monitoring can detect shifts in baseline sensitivity of pathogens and prevent postharvest fruit losses due to fungicide resistance. Site-specific or single site mode of action chemistries have been introduced that disrupt a metabolic process, and are more prone to develop resistance. Baseline information is routinely derived from a representative pathogen population before an active ingredient is introduced to market (Russell 2004). This allows researchers to establish a Minimum Inhibitory Concentration (MIC) or discriminatory dose for a specific active ingredient generated from an “unexposed” pathogen population based on mean and the range of EC50 values. The EC50 is defined as the concentration of fungicide that reduces fungal growth by 50 percent compared to growth on non-amended media (Secor and Rivera, 2012).

Research by two collaborating groups in Washington State and Maryland have independently determined a mean EC50 for unexposed, difenoconazole-sensitive Penicillium spp. populations (Ali and Amiri, 2018; Jurick et al., 2018). Mean EC50 values for 130 P. expansum isolates from Washington State was reported to be 0.17 ppm and was 0.16 ppm for 97 Penicillium spp. isolates largely obtained from Maryland and Pennsylvania. A discriminatory dose for monitoring difenoconazole resistance should be 1 ppm or higher and up to 5 ppm to detect truly resistant isolates. Baseline data, and corresponding discriminatory doses for MIC phenotyping, have been vital in monitoring fungicide resistance in Penicillium spp. populations and identifying fungicide-resistant blue and gray mold fungi (Li and Xiao 2008; Yan et al., 2014). Our laboratory has utilized published discriminatory doses for phenotyping fungicide resistant blue and gray mold pathogens with 0.5 ppm fludioxonil, 10.0 ppm Thiabendazole and 1.0 ppm pyrimethanil in agar-based Petri plates amended with technical grade chemicals (Li and Xiao 2008).

Recent Scientific Breakthroughs and Their Applications
The first genetic blueprint of the blue mold fungus was accomplished using Next Generation Sequencing Technology in the Penicillium expansum strain R19 (Yu et al., 2014). This isolate was obtained from decayed apple fruit in Pennsylvania commercial storage and shown to be highly aggressive when inoculated onto healthy apples. The P. expansum genome sequence is critical to understanding how this fungus develops resistance to various postharvest fungicides and has provided new clues about various infection strategies used by the fungus to decay apple fruit. Using sophisticated computer software analysis programs, Yu et al. determined that P. expansum R19 has 62 different secondary metabolic gene clusters and toxin biosynthetic pathways including one for patulin production. Hence, the fungus can produce a wide variety of chemicals/toxins/small molecules that may provide new uses in medicine and biotechnology as most have yet to be characterized. By sequencing and comparing different Penicillium spp. strains, genes involved in apple fruit decay, toxin production, and sexual recombination have been discovered (Julca et al., 2016; Wu et al, 2018; Yu et al., 2014). Even though the blue mold fungus has the genetic capacity to undergo sex, a definitive sexual stage for this fungus has not been observed in the laboratory or in nature. The practical impact of this discovery is that the fungus has the potential for genetic recombination, which can allow for movement of genes controlling decay, toxin secretion and/or fungicide resistance between different strains of the blue mold fungus. Hence, recombination could result in more fit strains capable of resisting multiple modes of action chemicals, and or become more aggressive resulting in increased control failures during storage.

Translating fundamental scientific information on the blue mold fungus is important and is envisioned to gain deeper insights into the genetic toolbox used by Penicillium spp. to cause decay in apple fruit. Once elucidated, the fungal tools that it uses can be exploited to develop controls that block decay from developing on apple fruit during storage. This is a similar approach to what is being done in cancer research to discover new drug targets to fight tumorigenesis, block metastatic development, and aid in early detection. Uncovering the genetic mechanisms of fungicide resistance will help tailor specific classes of new chemicals and natural products that provide durable control with lower likelihood for developing resistance. These discoveries will also enable the development of new detection tools that can be used not only by scientists, but producers as well, which would enable more timely detection of fungicide resistant isolates. Utilizing the latest molecular approaches such as CRISPR/Cas9-mediated genome editing, RNA interference, and single gene deletion strategies can be integrated to attack the fungus at multiple points that are critical for it to cause decay. These new tools for decay management, detection of fungicide resistant strains, and next generation chemistries to control postharvest rot will be a welcomed addition to the producers’ management scheme resulting in less decay, maintaining fruit quality over an extended period, and providing safer apple products devoid of mycotoxins. These breakthroughs will have wide impact and benefit our stakeholders and customers in industry, the scientific community, and the public at large.

Conclusions & Summary
Postharvest decay management currently follows an integrated pest management strategy that focuses on the pathogen including sensible fungicide application, implementation of sanitation methods in the packinghouse, and periodic fungicide resistance monitoring efforts. Current knowledge concerning utilization of different postharvest chemistries based on FRAC codes (Fungicide Resistance Action Committee) suggests that rotation is critical to maintain product efficacy. However, more research must be conducted to determine the impact of chemistry type on selection of fungicide resistant rot isolates, the frequency of rotation (e.g. yearly vs. throughout the season) and impact of preharvest fungicides to predispose fungi in the field for developing resistance in storage. In the meantime, cold chain management, bin sanitation and proper fungicide use will help keep decay at acceptable levels until new controls and methods are developed, refined, and implemented. For more information on specific postharvest decay control measures, please check out online resources provided by Cornell University, Penn State, and Washington State University Extension programs via the world-wide web.

Acknowledgements
Dr. Wayne M. Jurick II is the research plant pathologist and lead scientist on the project entitled “Development of Novel Tools to Manage Fungal Plant Pathogens that Cause Postharvest Decay of Pome Fruit to Reduce Food Waste” which is funded by United States Department of Agriculture (USDA)-Agricultural Research Service (ARS) National Programs 303 Plant Diseases. Financial support was also made possible through multiple competitive grants awarded to WMJII from the State Horticultural Association of Pennsylvania.

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