5

Role of Fungicide Use in Food Safety and Security

The fourth session of the workshop explored the role of fungicides in producing an adequate and safe food supply. Philip Taylor, training manager for Plantwise at the Centre for Agriculture and Bioscience International, moderated the session. Tim Brenneman, professor of plant pathology at the University of Georgia, discussed the role of fungicides in the peanut industry, the history of fungicide use and associated development of resistance, and the rise of resistant strains of Aspergillus. Pierce Paul, professor, plant disease epidemiologist, and state extension specialist in the Department of Plant Pathology at The Ohio State University, discussed the role of azole fungicides in disease control for field crops and factors that have affected fungicide use patterns in corn, wheat, and soybean production.

ROLE OF AZOLE FUNGICIDES IN SAFEGUARDING FOOD SAFETY AND SECURITY

Brenneman reviewed challenges in producing adequate food supplies, the role that fungicides play in meeting some of those challenges, and the different classes of agricultural fungicides. Within the context of the peanut industry, he discussed various products that have been introduced over the past 50 years, their efficacy against peanut leaf spot, and the resistance that has developed in response to most of these chemicals—particularly in Aspergillus.

Role of Fungicides in Food Production

Fungicides are necessary tools, among many others, that are needed to feed a growing world, said Brenneman. Food production must increase substantially to meet the needs of an expanding global population, with multiple analyses predicting that the volume of global food production may need to double by 2050 to meet the burgeoning demand. Unfortunately, maintaining food production levels is becoming increasingly difficult, making the goal of doubling the global food supply all the more daunting, he remarked. Among the host of barriers to increasing food production include soil degradation, urbanization, climate change, water depletion, scarcity of inputs such as fertilizers, and the effects of politics and armed conflicts. In addition to affecting food supply, some of these factors are influencing fungicide use. For example, many crops are grown in Texas and New Mexico, where fungicides are of lesser importance due to the dry climate. However, water supply in those states is decreasing, leading production to shift to the Southeast region of the United States where groundwater and rainfall are more abundant. Fungal diseases thrive under wetter conditions, so as production moves to more humid regions, the need for fungal disease control increases.

Fungicides are essential in keeping crops alive, Brenneman stated. In the absence of fungicide seed treatment, soil-borne diseases can prevent plants from sprouting (see Figure 5-1). Despite utilization of integrated pest management practices such as crop rotation, fungicides remain critical to food production, he remarked. Over the past century, the yield for peanut production per hectare in the United States has increased dramatically

to become the highest in the world. Although multiple factors have contributed toward this increase, the modern era of fungicide use has played a large role. Azoles have been instrumental in improving fungal disease control. Prior to their development, terraclor was used in the 1980s to address Athelia rolfsii (i.e., “white mold”). A granular material, terraclor was applied at 112 kilograms per hectare and resulted in 25–30 percent fungal disease control. In 1994, terraclor was replaced with tebuconazole, a liquid spray applied in quantities of 0.2 kilograms per hectare that achieved 75 percent control or better. In addition to treating Athelia rolfsii, tebuconazole controlled leaf spot and foliar diseases. The advent of azoles signified a new era of plant disease management.

Sterol Biosynthesis Inhibitors

Brenneman described sterol biosynthesis inhibitors (SBIs) as having a broad spectrum of activity and reducing some toxins such as deoxynivalenol (DON), a toxin that causes enormous losses in small grains. Featuring varying degrees of systemicity and post-infection activity, SBIs have several advantages over previous protectant fungicides. Some SBIs are relatively inexpensive, particularly older products such as tebuconazole that are available in generic versions. However, he noted that the low price of SBIs could potentially contribute to overuse. Labeled for use on a wide variety of crops, SBIs represent approximately one-third of the global fungicide market. Thus, this class of fungicides plays a significant role in disease control in the food production system. Four subclasses of SBIs include demethylation inhibitors (DMIs), amines or morpholines, hydroxyanilides, and squaleneepoxidase inhibitors. The largest subclass, DMIs include 36 different fungicides, including azoles. Resistance to DMIs is not uncommon, although it seldom causes a complete loss of efficacy, as has happened with other fungicides such as quinone outside inhibitors (QoIs) or the benzimidazoles. A gradual loss of efficacy can occur that is associated with the quantitative or stepwise polygenic resistance inherent with DMIs. This class can also experience backshifts due to fitness loss, which can be mediated to some degree by temporarily pulling the fungicide from use. Multiple documented mechanisms of resistance create a scenario that complicates the ability to determine which mechanism is at play. A newer generation of DMIs is often more active than older fungicides due to the quantitative shift. Therefore, despite these newer DMIs being subject to the same resistance mechanisms, they have a higher level of inherent activity. Brenneman described DMIs as a critical fungicide class for many production systems that features long-lived chemistry.

The Rise and Fall of Fungicides for Peanut Leaf Spots

Brenneman provided an overview of the waves of fungicides used to treat peanut leaf spots over the past 50 years. The 1970s saw the introduction of benzimidazoles and chlorothalonil. Benomyl, a single-site mode-of-action benzimidazole, was only effective for approximately 3 years before widespread use and single-site mutations led to virtual immunity in leaf spot. In contrast, chlorothalonil has been in use for 50 years with no cases of resistance. This multisite fungicide has maintained integrity and has helped address resistance in other fungicide classes. In the 1980s, several DMIs became available such as propiconazole, tebuconazole, and fenbuconazole. These single-site fungicides worked for about a decade before resistance began appearing. Brenneman highlighted that the introduction of tebuconazole was a game changer in eliminating leaf spot almost entirely. However, within 10 years, resistance levels were such that some peanut crops treated with tebuconazole were still nearly defoliated from the disease. He noted that tebuconazole continues to be effective against some soil-borne pathogens. In the 1990s, single-site QoIs such as azoxystrobin and kresoxim-methyl became available. Once again, resistance developed within about 10 years, and efficacy of this class was essentially lost for leaf spot. Succinate dehydrogenase inhibitor (SDHI) fungicides were introduced 10–15 years ago, and some signs of resistance are beginning to show within this class as well. Brenneman added that chlorothalonil has been used in combination with all the other classes in an attempt to manage the resistance, yet resistance has developed nonetheless.

Brenneman highlighted data that indicate that adding micronized sulfur to DMI fungicides results in synergistic effects on peach scab and peanut leaf spot (Culbreath et al., 2019; Schnabel and Layne, 2004). In the study, untreated peanut crops experienced nearly 100 percent defoliation. Tebuconazole, once highly effective before resistance diminished its effect, only decreased defoliation to 70–80 percent. Micronized sulfur used independently reduced defoliation to 40–50 percent. When tebuconazole and micronized sulfur were used in conjunction, a synergy took effect and achieved a defoliation rate of less than 10 percent. Thus, adding micronized sulfur to the fungicide helps overcome the effects of resistance. He added that similar synergistic effects have been demonstrated with QoIs and SDHIs.

Aspergillus Resistance in Peanuts

Climate change-related heat and drought have contributed to a rise in Aspergillus in peanut crops, as the fungus appears to thrive in these conditions, said Brenneman. Aspergillus flavus (A. flavus) and Aspergillus niger are the seed-borne species that pose the most substantial problems

in the peanut industry. Both species have developed resistance to QoI fungicides, requiring farmers to change seed treatment regimes. Previously, azoxystrobin was a main component of seed treatment, but in 2020, the peanut industry shifted to an azole-based regime comprised of ipconazole, SDHIs, and prothioconazole to compensate for QoI resistance.

The QoI fungicides inhibit cellular respiration through cytochrome b, Brenneman explained (Fernández-Ortuño et al., 2010). They have a known high-risk single-site mode of action, and multiple point mutations confer different levels of resistance from partial to immunity. He and his colleagues have identified two mutations in the mitochondrial cytochrome b gene that confer A. flavus resistance (Ali et al., 2021). A wild-type allele that has not mutated has an average EC50 value (i.e., the concentration of a drug that gives half-maximal response) close to 0, signifying high sensitivity to QoI chemistry. In contrast, alleles associated with the F129L mutation phenotype had EC50 values of 50 and the G143A mutation phenotype had EC50 values well over 100. Brenneman emphasized the importance of phenotyping for DMI resistance due to the large range of phenotypes and the potential combinations of resistance mechanisms in response to DMIs. Resistance factors can vary substantially, thus phenotyping is required to fully capture resistance levels.

Brenneman remarked that while DMI fungicides are losing effectiveness, they continue to make valuable contributions to food production. Resistance is a pressing threat to all fungicide chemistries: that is, each time a class of fungicide is lost to resistance the difficulty in preserving remaining classes increases. Thus, the more modes of action the agriculture industry can utilize, the more effective antifungal efforts are likely to be. The loss of older multisite products to resistance could dramatically accelerate the lack of remaining fungicide tools. Acknowledging the scrutiny placed on chlorothalonil, he stated that losing this product and using single-site modes-of-action fungicides alone would increase challenges in addressing fungicide resistance. Therefore, new approaches to plant disease management are needed, with molecular approaches like RNA interference holding promise in reducing selection pressure on fungicides and prolonging the life of existing fungicides.

AZOLE USE FOR FOOD SAFETY AND SECURITY

Paul provided a field crops perspective on the use and importance of azole fungicides for disease control and, consequently, for food safety and security. He discussed the factors that created significant shifts in fungicide use patterns in field crops over the past 20 years and described the role of azoles in addressing Fusarium head blight. The varied fungicide formulations are classified into three chemical groups according to mode of action:

QoIs, DMIs, and SDHIs.¹ DMI fungicides (i.e., azoles) and QoIs are the predominant products in current use. Most field crop fungicides are used for above-ground diseases—specifically foliar diseases—although some products also offer seed and seedling disease control capability. Paul noted that fungicides are particularly important in controlling rust diseases in wheat, corn, and soybeans. These products offer effective control of several diseases that affect both yield and quality, making them an integral part of disease management programs for field crops.

Recent Shifts in Field Crop Fungicide Use Patterns

A major shift in fungicide application and use patterns in field crops occurred in the mid-2000s. At that time, fungicide use became widespread in response to multiple factors, said Paul. Claims that plant health benefited from use of fungicide—even when disease was absent or at low levels—contributed to a change in product labels. When limited control environment studies indicated that QoIs affect crop physiology such that yields could increase even in the absence of disease, some labels began listing a plant health benefit. This led some people to believe that spraying fungicide on healthy plants would increase yield, thus fueling the widespread use of fungicides.

Additionally, grain prices increased and modern hybrids of corn saw higher yield potential during this period. Historically, fungicide use in field crops was cost prohibitive because prices were not high enough to offset application costs. With the increase in both prices and yield potential, expenditures on fungicides became more appealing. This particularly contributed to fungicide use pattern changes within the corn belt. Furthermore, major disease outbreaks and threats occurred during this time, such as soybean rust. Several major azole fungicides were limited to certain crops in their labeling, but the soybean rust outbreak led to section 18 exemptions under the Federal Insecticide, Fungicide and Rodenticide Act,² which authorizes the U.S. Environmental Protection Agency to allow unregistered uses of pesticides to address emergency conditions. Several azoles attained full labels and registration via this avenue. Paul noted that azoles also gained labeling for other field crops in response to farmers stockpiling fungicides for soybean rust and then needing ways to use their surplus. Thus, the soybean rust outbreak led to increased azole use in field crops other than soybeans.

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¹ More information about fungicides and efficacy levels is available at https://cropprotectionnetwork.org/publications?collections=%5B%22Publications%22%5D (accessed August 7, 2022).

² Federal Insecticide, Fungicide, and Rodenticide Act of 1947, Public Law 80-104, 80th Cong., 1st sess. (June 25, 1947).

The mid-2000s also saw a boom in the ethanol industry with the establishment of numerous ethanol plants. The demand generated by ethanol plants led farmers to plant more acres of corn. Paul described that the production of continuous corn also increased in an effort to meet demand; this shift from crop rotation caused more disease. This time period coincided with another change in production practices that also contributed to fungicide use: the practice of conservation tillage to help reduce erosion. These various factors combined to cause a sizable increase in fungicide use in field crops in the mid-2000s. Paul added that in the years since, fungicide use has dropped considerably overall, but remains widely used in field crops.

Despite the considerable use of fungicides in field crops, the production of corn, wheat, and soybeans does not rely as heavily on these chemicals as do other agricultural production systems, said Paul. Typically, a single application is sufficient for effective disease control for field crops. He noted that recommendations for a second application have been made for some cases of southern rust and tar spot diseases. However, in most cases—provided the application is timed correctly—a second application is not required. Moreover, the value of the crop often does not justify a second application, as the benefit is insufficient to offset the application cost. Label restrictions also contribute to lower use of fungicides in field crops than in other agricultural industries. The majority of field crop diseases develop in the early reproductive stages but applying a second application often infringes on legal pre-harvest intervals. Paul stated that these factors combine to prevent excessive use of fungicides in field crops in comparison to other production systems.

Role of Azoles in Field Crop Production

Paul explained that azoles are the main ingredient in most types of field crop fungicides. Of the 24 fungicides used on corn, 19 contain an azole active ingredient. In soybeans, 26 of the 34 fungicides are azole-based, and the proportion is even higher for wheat, with 15 of 18 fungicides containing azoles.³ Across these three crops, seven core primary azoles are featured in the various azole-based fungicides: flutriafol, propiconazole, prothioconazole, tebuconazole, tetraconazole, cyproconazole, and metconazole. Paul clarified that the 26 azole-based fungicides used on soybeans comprise 26 different combinations of 7 azoles, not 26 different azole active ingredients. Among the most effective fungicides against economically important diseases, azoles are effective against leaf spots, blotches, and rusts, the latter being some of

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³ More information about azole-based fungicides used in field crops is available at https://cropprotectionnetwork.org/publications?collections=%5B%22Publications%22%5D (accessed August 7, 2022).

the most damaging diseases to field crops. Azoles are used in rotation or in combination with QoIs and SDHIs as a fungicide resistance management strategy and to increase the spectrum of activity. Additionally, azoles are the only control option for some diseases such as FHB (Fusarium head blight) and Gibberella ear rot and the mycotoxins associated with these diseases.

Fusarium Head Blight

Caused by Fusarium graminearum, FHB causes bleached, discolored spikes in wheat that lead to mycotoxin grain contamination. Paul stated that this fungal disease poses a major food safety concern due to features of the mycotoxins. FHB is associated with several toxins, including DON. Both water soluble and heat stable, DON spreads easily and persists after being cooked or baked. Moreover, DON can conjugate with other compounds, enabling it to be masked and hide undetected until it enters an animal system, at which point it can be unconjugated to release the active DON toxin again. Azoles are one of the most effective fungicides for reducing both FHB and associated mycotoxins.

Integrated management guidelines for FHB include using the most resistant variety of grain, crop rotation, tillage, and fungicide. Using genetically resistant grain varieties is not effective independent of other measures, because no variety is immune and some of the most resistant varieties offer lower yields. Tillage and crop rotation are inadequate in and of themselves due to the ability of spores to travel easily. Paul stated that fungicide is a necessary component of an integrated management program that effectively reduces FHB and the mycotoxin contamination of grain. Several azoles are considered industry standards for FHB, including triazole, tebuconazole, prothioconazole, and metconazole. Proline and Caramba are products that use individual azoles, and Prosaro and Sphaerex utilize azole combinations. Increasing fungicide resistance has led to combination products comprised of azoles and SDHIs. Efficacy data indicate that azole-based fungicides generate more than a 50 percent reduction in both disease and mycotoxin contamination of grain (Edwards and Godley, 2010). Newer combinations of fungicides utilizing azoles and SBHIs achieve similar results, although it is notable that the new active ingredients have not been found to be more effective than existing active ingredients.

Azole-based fungicides remain the most effective tool in reducing FHB and DON in wheat, said Paul. In fact, QoIs used in isolation on field crops have been found to increase DON levels in grain rather than reduce them. Five different QoI products were found to increase DON levels from 6–18 percent above untreated wheat. In contrast, azoles maintain grain yield and quality, reduce food safety concerns, and benefit numerous industries including livestock, milling, baking, brewing, and ethanol.

DISCUSSION

Quinone Outside Inhibitors and Mycotoxin Levels

Given that a fungicide with even limited activity would be expected to reduce mycotoxin production of the fungus, Taylor asked Paul to expound on how QoIs were found to increase DON production. Paul affirmed that plants treated with QoIs did have higher average levels of DON than plants that are untreated or that are treated with azoles. In some cases, the QoIs reduce visual symptoms and disease levels of FHB, yet the mycotoxin contamination levels in grain simultaneously increase. A hypothesis for this phenomenon is that QoIs selectively control non-mycotoxin-producing fungi, thus leaving the mycotoxin-producing fungi to thrive in the wheat spikes and produce higher levels of mycotoxin. Additionally, QoI fungicides keep plants greener for longer, which increases the likelihood of higher levels of mycotoxin accumulating over time.

Fungicide-Related Fitness Cost

Taylor asked whether a pathogen experiences any fitness costs when it becomes resistant to fungicides. For instance, does the pathogen become less virulent or sporulate less? Paul replied that he was unaware of any reported fitness cost associated with resistance. Brenneman stated that fitness cost varies with fungicide class: fungi resistant to benlate experienced little if any fitness cost; resistance to triazoles is known to have some fitness cost; and fungi resistant to triphenyltin has shown clear fitness cost, with high levels of resistance observed 1 year and resistance levels subsiding considerably the following year. This exemplifies how pulling a fungicide out of the system can reset the timetable on efficacy loss.

Micronized Sulfur

Replying to a query about whether sulfur must be micronized in order to cause the synergistic effect of reducing resistance, Brenneman remarked that research conducted by Albert Culbreath, his colleague at the University of Georgia, indicated that traditional, larger-particle size sulfurs do not achieve the same effect (Culbreath et al., 2019). He added that micronized sulfur’s ability to boost fungicide efficacy is significant in helping to maintain efficacy of some at-risk chemistries; this effect was seen in QoIs, SDHIs, and the DMIs.

Resistance to Recent Azoles

Taylor asked whether any resistance has been identified in the newer azoles—namely, prothioconazole and mefentrifluconazole—and whether they are expected to remain effective for a longer period given that resistance is already in the system. Paul replied that some gradual reduction in sensitivity has been found for FHB in the wheat system, but he would not necessarily characterize this as resistance. However, he noted that evidence of resistance in FHB as well as other diseases has been found in Europe, where these products are used more frequently than in the United States. A gradual eroding of sensitivity to these fungicides is evident, especially in comparison to tebuconazole and metconazole, which have older active ingredients than prothioconazole.

Considerations in Fungicide Blends and Concentration Rates

In terms of utilizing multiple fungicides, Taylor asked whether mixing fungicide blends or rotating the fungicide used between seasons or applications is more likely to prevent resistance. Brenneman remarked that this topic is debated among plant pathologists. He stated his view that the best method varies with the system. In small grains that receive only one or two applications, blended combinations play a substantial role in prevention. In peanuts or pecans, a season-long strategy that utilizes a single available active ingredient at a time provides flexibility; if one of the products meets resistance, other options remain available, whereas a blend might involve all available options in the first application. Brenneman added that the process of mixing fungicides can be complicated. If all the fungicides are used at full rate, this approach becomes expensive. Using high rates can also induce a gradual shift in sensitivity, and selection for higher and higher rates may take place. On the other hand, if rates are reduced to sublethal levels and the product becomes ineffective, that too causes problems. The ideal mixture cuts the rate while maintaining efficacy via the combined modes of action.⁴

Paul noted the role of pre-mixes, in which chemical companies create the mixtures. These pre-mixes use lower rates of the two active ingredients in comparison to the rate of each ingredient in a single-active-ingredient product. Assuming the companies have carried out adequate research in establishing rates for these mixtures, the products should still be effective at these lower rates. He added that most of the blends used are pre-mixed, but on infrequent occasions, products have been mixed in the field.

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⁴ This topic was further discussed at the end of the day; see the discussion section at the end of Chapter 6.

Noting that limited literacy and numeracy skills among some agricultural workers can lead to inadvertent divergence from fungicide label recommendations, Jeff LeJeune, safety officer in the Food Systems and Food Safety Division, Food and Agriculture Organization at the United Nations, asked about the relationship between the misuse of fungicide concentration levels and excess application. Paul replied that the general understanding of sublethal doses is that they increase fungicide resistance by exposing organisms without killing them. The surviving subpopulations then develop resistance, which is passed on and increases with each subsequent generation. When fungicides are developed, various concentration rates are tested, informing recommendations about the rate that is lethal to the fungus or spores. Paul stated that he does not know what effect higher-than-recommended concentrations have on resistance. Models have indicated that the general understanding of exposure to sublethal doses does not hold with every pathogen or system. For example, research reveals that in some situations higher dose rates of fungicide can be more problematic than half rates (van den Bosch et al., 2018).

LeJeune asked whether the use of fungicides at lower-than-recommended concentrations, due to cost considerations, is known to affect resistance. Paul remarked that he does not have specific data on this dynamic, but use patterns suggest waves of outbreaks associated with people veering from recommended doses as a cost-saving measure and then returning to label dosing guidelines when disease occurs. For example, in the mid-2000s, fungicide was commonly used at the half dose rate during earlier growth stages, based on the assumption that a half dose would provide a smaller plant with adequate coverage. Once plants grew in size, the full dose rate was used. After several years of this practice, it fell out of favor not because of resistance, but because it was found to be ineffective at increasing disease control and yield. Paul added that research of such practices in real-world situations is insufficient to determine exact repercussions of dosing deviations, but simulation models demonstrate which effects are likely or unlikely.

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