Impact of fungicide application to control T-2 and HT-2 toxin contamination and related Fusarium sporotrichioides and F. langsethiae producing species in durum wheat

Highlights

• In field efficacy of azole fungicides on Fusarium langsethiae/F. sporotrichioides.

• T-2 and HT-2 toxins 70–80% reduced after fungicide application.

• With artificial infection, prothioconazole was the most effective among azoles.

• Azoxystrobin was the less effective in containing infections.

• At high concentrations of both species on wheat, F. sporotrichioides is favored.

Abstract

Fusarium Head Blight (FHB) is the main devastating disease of wheat worldwide. Most of the studies are addressed to control the main fungal agent F. graminearum. However, the FHB species composition is wider and influenced by meteorological and agronomic factors, depending on geographical region and, more recently, subjected to the ongoing climate change. Many strategies to control FHB agents in field conditions have been studied. Among these strategies, the fungicide application proved to be among one of the most effective. To date, very poor investigations were carried out to evaluate the effects of fungicides in wheat against F. sporotrichioides and F. langsethiae, the species associated to FHB that produce type A trichothecenes, such as T-2 and HT-2 toxins.

The effectiveness of the triazoles, prothioconazole and metconazole, and the strobirulin azoxystrobin, alone and in combination with tebuconazole, was tested in three field trials, two locations and in two growing seasons, in Italy, against F. sporotrichioides and F. langsethiae species, to evaluate their impact on the T-2 + HT-2 contamination in wheat. The trials were carried out by comparing the different fungicide effects, under natural infection and on artificially infected wheat plants.

FHB incidence and severity, significantly different between the two geographical locations, were effectively reduced by fungicide application. Similarly, F. sporotrichioides and the positively correlated T-2 and HT-2 amounts, were effectively reduced up to a range of 53–91% under natural infection and 30–70% under artificial infection, depending on the active ingredient. On the contrary, F. langsethiae was effectively reduced by fungicides only in natural conditions, while it appears to be insensitive to fungicide treatments under artificial infection trials. In addition, the artificial inoculation with this species failed to achieve high level of infection. Moreover, qPCR was proved to be among the needful methods to detect F. langsethiae, whose isolation failed by classic mycological method.

In this first study on fungicide effectiveness against F. sporotrichioides, F. langsethiae and T-2+HT-2 contamination in wheat plants in field trials, we have demonstrated that triazole compounds are able to mitigate the possible contamination of type A trichothecenes and their producing species. Among the tested fungicides, prothioconazole demonstrated to be the most effective against both fungal species and under all the experimental conditions. On the other hand, azoxystrobin showed to be less effective in presence of high fungal infection.

Introduction

Fusarium Head Blight (FHB) is the most devastating disease of wheat crop worldwide, causing yield reduction and contamination of mycotoxins, secondary metabolites that cause adverse effects on humans and animals, often occurring as a result of Fusarium infections (Desjardins, 2006). Several Fusarium species and Michrodochium nivale are causal agents of FHB, with F. graminearum, F. culmorum, F. avenaceum, F. poae, F. sporotrichioides, F. equiseti, F. langsethiae, and F. tricinctum, the most reported Fusarium species associated with the disease (Parry et al., 1995; Bottalico and Perrone, 2002). The species composition of the FHB complex changes depending on the climatic and environmental conditions (Wegulo et al., 2015). Thus, the predominance and frequency of individual Fusarium species can vary in different geographical areas (Xu et al., 2005). Usually, F. culmorum and F. avenaceum are more frequent in cool and wet conditions, F. graminearum dominates in temperate areas with humid climatic environment, F. poae is more associated with warmer and drier conditions (Doohan et al., 2003; Xu et al., 2008).

A correct identification is extremely important since Fusarium genus includes many species that produce a wide range of mycotoxins, and each species has its own mycotoxin profile. Indeed, F. avenaceum and F. culmorum are significant or even dominant Fusarium species in cereal grains in most parts of Northern Europe (Levitin, 2004; Kosiak et al., 2003; Yli-Mattila et al., 2008; Nielsen et al., 2011); F. sporotrichiodes has been reported in prevalence in Eastern and Northern Europe (Gagkaeva et al., 2019); F. graminearum is the most important Fusarium species in Central Europe (Xu et al., 2005; Gagkaeva and Yli-Mattila, 2004; Yli-Mattila, 2010). However, changes in climate are leading to dramatically influence the species composition in Europe. Thus, in recent years, F. graminearum has been spreading to the North of Europe and it has been replacing F. culmorum (Waalwijk et al., 2003; Fredlund et al., 2010; Yli-Mattila et al., 2013). In addition, in recent years, also an increase of the occurrence of F. poae among the species associated to FHB has been observed (Klix et al., 2008; Xu et al., 2008). Other species of minor frequency in the FHB complex, such as F. sporotrichioides, F. crookwellense, F. equiseti, F. tricinctum and F. langsethiae, can play a significant role when weather conditions are not favorable for the growth of the main species (Mielniczuk and Skwaryło-Bednarz, 2020). Although the presence of the main FHB agents has occurred often in Italy, in the last two decades a continuous increase of F. poae has been recorded (Shah et al., 2005; Pancaldi et al., 2010). However, in the past, the presence of F. poae was overestimated because it included F. langsethiae, whose spore morphology is indistinguishable (Torp and Nirenberg, 2004; Yli-Mattila et al., 2004; Knutsen et al., 2004). The distinction between these two species is important, since F. langsethiae produces T-2 and HT-2, the most harmful Fusarium mycotoxins, while F. poae does not (Proctor et al., 2009; Witte et al., 2021). Moreover, F. langsethiae infections on plants are mostly symptomless, which make them difficult to detect. Another T-2 and HT-2 producing species, F. sporotrichioides, is morphologically also very closely related to F. poae and F. langsethiae, and often co-occurs in the wheat grains. Although the isolation of F. langsethiae and F. sporotrichioides from grains has been demonstrated to be difficult (Imathiu et al., 2013a; Gavrilova et al., 2017; Edwards et al., 2012), causing underestimation of both species, their increase has been observed in recent years in Italy (Infantino et al., 2015; Morcia et al., 2016; Beccari et al., 2020; Haidukowski et al., submitted). In particular, in Italy, differences in species compositions among geographical areas were assessed and FHB infections were reported to increase from Southern to Northern Italy, with significant link to the amount of precipitations during wheat anthesis (Pancaldi et al., 2010; Covarelli et al., 2015).

Durum wheat, that is more susceptible to FHB than common wheat, is widely cultivated in Italy. Thus, the risk of mycotoxin accumulation in kernels is of particular concern when this crop is cultivated in geographic areas where the environmental conditions lead to the disease, such as Northern and Central regions (Xu et al., 2008). To date, F. sporotrichioides and F. lagsethiae have been poorly investigated, and only few studies were focused on their occurrence and ecology.

Both species are the main producers of T-2 and HT-2 toxins, two type A trichothecenes very harmful for human and animal health, and the most toxic among trichothecenes, since they inhibit DNA, RNA and protein synthesis and induce DNA fragmentation, causing acute or chronic intoxication of humans and animals. The European Commission adopted Recommendation for these mycotoxins, with the indicative level of 100 μg/kg for the sum of T-2 and HT-2 in unprocessed durum wheat (Commission Recommendation, 2013/165/EU). However, no legislative limits are established, although they are currently under consideration.

Under field conditions, together with environmental conditions, the spreading of species involved in FHB and their ability to produce mycotoxins, is influenced by many agronomic factors, such as tillage, cultivar selection, and use of fungicides.

Since information on F. sporotrichioides and F. langsethiae with respect to their epidemiology, environmental requirements for infection and toxin production, and influence of agronomic factors, are poor, compared those on F. graminearum, the strategies for their control management have not been assessed yet.

Kokkonen et al. (2012) and Nazari et al. (2019) studied the interaction of Fusarium species producing trichothecenes type A with temperature and humidity requirements in vitro and in vivo, respectively. Moreover, Nazari et al. (2019) investigated the relationship between plant growth stage, time of infection and the colonization and mycotoxin production of F. sporotrichioides, F. langsethiae and F. poae. Although weather conditions at flowering is well known as one of the most important factors for infection of FHB species (Parry et al., 1995; Edwards and Jennings, 2018), including F. sporotrichioides, the same finding was not evident for F. langsethiae (Nazari et al., 2019). The different behaviour of these fungal species, arise the need of more information about for control strategies, including fungicide application. Indeed, although preventive agronomic factors, as crop rotation, soil tillage and use of resistant varieties are important strategies to minimize the fungal and mycotoxin contamination in the field, in climatic conditions favorable to fungal infection (Blandino et al., 2012), these measures could be integrated by a direct control through fungicide application to increase the efficacy of the control (Mesterházy et al., 2003). In particular, fungicide application at flowering is the most effective practice in temperate areas (Ransom and McMullen, 2008). However, the selection of the optimal timing of the application is crucial (Wegulo et al., 2015).

Fungicide application against FHB agents has been focused mostly on F. graminearum and deoxynivalenol (DON) reduction. The most effective fungicides used in controlling FHB are demethylation inhibitors (DMIs). Azole fungicides, such as tebuconazole, metconazole and prothioconazole, have proven to provide the most effective control of FHB infections and DON contamination in wheat (Paul et al., 2008), although none of them is sufficient alone (Dweba et al., 2017). Tebuconazole and prothioconazole applied at anthesis reduced FHB severity and DON content up to 90% in field trials (Haidukowski et al., 2005, 2012; Paul et al., 2008). However, these fungicides induce resistance in Fusarium, due to repeated applications (Paul et al., 2018). Therefore, mixtures with different other fungicides have been suggested, according with the integrated pest management (IPM) strategies.

On the other hand, triazole efficacy against the Fusarium species producing T-2 and HT-2, F. langsethiae and F. sporotrichiodes, was poorly, if ever, investigated.

The aim of this study was to assess for the first time the efficacy of triazole fungicides, also in combination with a strobirulin fungicide, on durum wheat, against F. sporotrichiodes and F. langsethiae species and related T-2 and HT-2 contamination on wheat, under both naturally and artificially-infected field conditions. In particular, four different fungicides, containing prothioconazole, metconazole, azoxystrobin and a mixture of azoxystrobin and tebuconazole, were tested on durum wheat grown in three fields experiments in Italy, and in two different growing seasons.