Pathogens, Vol. 11, Pages 1427: Biofumigation for the Management of Fusarium graminearum in a Wheat-Maize Rotation

1. IntroductionWheat is one of the most important agricultural crops globally. In terms of dietary intake, it is the most important staple commodity [1] and the most widely grown crop with more than 219 million ha grown worldwide in 2020 [2]. Maize is the third leading staple after rice and the most produced crop in the world [1]. An average global production of over 1 billion t of maize has been reported for the eight-year period (2013–2020) [2]. In 2020, the area under maize cultivation in the EU was 9 million ha producing 67 million t [2]. A major disease problem in wheat and maize is caused by the fungal pathogen Fusarium graminearum which causes Fusarium head blight in wheat and ear and stalk rot in maize [3,4]. Fusarium graminearum infection causes shrivelled kernels, poor grain quality and mycotoxin contamination. The harvested grains which may appear normal and healthy can still be contaminated with mycotoxins [5]. The mycotoxins of most concern produced by F. graminearum are deoxynivalenol (DON) and zearalenone (ZON). Deoxynivalenol induces vomiting, anorexia and reduced food intake in humans and animals. It also has hepatotoxic, immunotoxic and neurovirulent effects [6]. In contrast, ZON mainly has estrogenic effects in humans and animals, causing reproductive and fertility disorders and premature puberty [7]. Additionally, ZON may also cause hematotoxicity, genotoxicity, hepatotoxicity and immunotoxicity [8]. In addition to health consequences, these mycotoxins are responsible for significant economic losses. In the US, Fusarium head blight resulted in yield losses worth $1.176 billion in 2015/16 [9]. For consumer protection, the European Commission has set legislative limits for ZON at 100 µg kg−1 and for DON at 1250 µg kg−1 in wheat. In maize the limits are set at 350 µg kg−1 and 1750 µg kg−1 for ZON and DON, respectively [10]. Contamination exceeding legal limits of DON and ZON were reported in 13% and 29% of wheat samples, respectively, in England in 2008 [11]. In Croatia, 50% and 28% samples of maize collected in 2010 contained DON and ZON, respectively, at concentrations above the legal limits [12]. Results from a recent survey conducted in Luxembourg [13] showed that concentrations of DON above the legal limits were detected in 5% wheat samples collected during a 12-year period (2007–2018). In the same study, 15% of wheat samples from 2018 contained DON levels above legal limits. There are different factors that are important for the establishment and development of Fusarium head blight. These include humid weather conditions during anthesis [14] and density of residues from previous crops, affecting the severity of the disease and DON contamination [14]. Maize as a preceding crop is known to be an important risk factor for Fusarium head blight in the subsequent wheat [15] as large quantities of infected residues that serve as substrate for F. graminearum remain after harvest [16]. Fusarium graminearum survives as mycelium on crop residues and produces ascospores (sexual) and conidia (asexual). These spores and hyphal fragments, air-dispersed and water-splashed, serve as source of inoculum in the subsequent cereal crop [17,18].Currently, F. graminearum is managed through triazole fungicides such as prothioconazole and tebuconazole, however due to the endocrine-disrupting properties of these fungicides, serious concerns have been raised [19]. Due to these safety concerns, their limited effectiveness and high selection pressure for fungicide resistance [20], there is a need to explore an alternative control strategy. Biofumigation as a crop protection strategy has recently gained interest. This practice involves growing short term brassica crops, followed by maceration of the plant tissue and rapid incorporation into the soil. Inhibitory volatile substances, particularly isothiocyanates (ITC) are produced as a result of damage to brassica plant tissue causing suppression of soil-borne pests and diseases [21]. The suppressive effect of biofumigation is generally attributed to the toxicity of isothiocyanate (ITC) which is produced as a result of glucosinolate (GSL) hydrolysis by myrosinase enzyme upon plant tissue disruption. However, the application of green manure also increases the organic matter content in the soil which supports soil saprophytes, thus enhancing their competition and antagonism effects. Additionally, toxic compounds released during the decomposition of the organic matter may contribute to the suppressive activity [22,23,24].Whilst biofumigation has attracted significant interest, research on its potential application for reducing the inoculum of Fusarium species affecting cereals is scarce. Previously, fungal pathogens of potato were suppressed using biofumigation in the glasshouse and in the field [25]. Incorporation of Brassica juncea was shown to reduce disease incidence and severity of powdery scab (Spongospora subterranea) and common scab (Streptomyces scabies) by 40% and less than 20%, respectively. Recently, Drakopoulos et al. [26] applied the mulch of Sinapis alba and B. juncea, in a cut-and-carry approach, to F. graminearum-infected wheat plots; Fusarium head blight incidence was significantly reduced by 58% in the first year by S. alba and 18% by B. juncea in the second year, in field experiments performed over two years.Following encouraging results from studies conducted under laboratory conditions [27,28], a field experiment was undertaken to investigate the potential of incorporation of brassica crops on suppressing F. graminearum inoculum in a wheat-maize rotation. 4. DiscussionIn the present two-year study, biofumigation was not effective in suppressing F. graminearum in the soil and in the following maize crop. Following incorporation of brassicas, F. graminearum infection in maize was not significantly lower compared to control (fallow) plots. This is in contrast to results from previous work in the laboratory [27,28] indicating brassicas could have a suppressive effect on F. graminearum under field conditions.Biofumigant brassicas have been effective in suppressing soil-borne pathogens in studies conducted elsewhere. For example, Subbarao et al. [40] reported suppression of Verticillium wilt in cauliflower by broccoli residue incorporation in comparison to metam sodium, chloropicrin and other control treatments. The soil population of Verticillium dahliae microsclerotia was reduced by 50–75% following incorporation of broccoli, compared to pre-treatment levels. Drakopoulos et al. [26] reported 40–50% DON reduction in wheat flour by using B. juncea and S. alba mulch in field experiments. While these studies have demonstrated the effectiveness of biofumigation in suppressing disease, lack of disease control has also been reported. In a field experiment [41], where F. oxysporum, Rhizoctonia solani and V. dahliae inoculum were buried in soil previously amended with broccoli at 34–38 t ha−1 (fresh weight), no suppression was recorded. Similarly, in another study [42], incorporation of B. juncea and B. napus in field was not effective in suppressing F. oxysporum or Pythium spp. populations in soil.There could be a number of factors that might have impacted the efficacy of biofumigation in the present study. Soil temperature has a significant impact on ITC production [43]. Previously, soil amended with cabbage residues was analysed for volatile production [44]. It was reported that the concentration of volatiles in the headspace were higher in heated, amended soils than in non-heated amended soils. In the present study, when the plants were incorporated in November, mean soil temperatures at ~10 cm deep were 7.7 °C which might not have been high enough to favour effective ITC production. Moreover, due to dry weather conditions, ITC production might not have been sufficient. The average rainfall for November 2018 was 36 mm which was half the amount compared to the average rainfall for November (72 mm) for the previous five-year period (2013–2017) according to the data from nearest weather station at Shawbury, UK [45]. Presence of sufficient moisture is important to enable myrosinase activity for production of GSL hydrolysis products [46]. In a field study conducted by Matthiessen et al. [47], addition of 42 mm of water to B. juncea plant material resulted in a 7- to 10-fold increase in ITC concentrations in soil compared to where no water was added. Wang and Mazzola [48] amended soil with B. juncea and S. alba seed meal in jars and evaluated AITC emission in the headspace. They reported that AITC production elevated (~0.05–0.265 µg g−1 soil) with an increase in soil temperature from 10 °C to 30 °C and increase in moisture level from −1000 kPa to −40 kPa. Another factor that might have affected the activity of ITC could be the organic matter content in the soil. Gimsing et al. [49] demonstrated that organic matter content is the main sorbent of ITC. Organic matter in arable soils is usually at 2–4% [50] compared to which the organic matter content in soil at the field experiment site was higher at 6%. Sorption of ITC to the organic matter in soil might have resulted in reduced activity of ITC.In a study investigating the ITC-release potential of brassicas under field conditions, R. sativus, at a seed rate of 20 kg ha−1 produced a biomass of 71–74 t ha−1 and an estimated 31–45 mmol m−2 GSL [51]. Conversely in the present study, at a similar seed rate, R. sativus produced similar biomass (74–79 t ha−1) but the expected GSL concentration in the field was much lower (9–10 mmol m−2). The difference in GSL concentrations could be due to difference in soil conditions such as temperature and moisture. The predominant GSL in the tissue of B. juncea was sinigrin as reported previously [51,52]. Sinigrin concentration estimated for B. juncea (above-ground) was 11 mmol m−2. In comparison, Doheny-Adams et al. [51] found a higher concentration of 16–24 mmol m−2 under field conditions. Isothiocyanate-release potential is dependent on GSL concentrations, which were sub-optimal in the present study. In contrast, total GSL concentrations as high as 93, 69 and 61 µmol g−1 biomass have been detected previously in B. juncea, R. sativus and E. sativa, respectively, under field conditions [33].Biofumigants grown during summer conditions are exposed to higher UV intensity, longer daylight hours and higher temperatures. These factors are known to increase the production of GSL in brassica tissues [53]. Ngala et al. [33] showed how summer grown brassica crops (B. juncea, E. sativa and R. sativus) produced higher concentrations of GSL in the summer when compared to being overwintered. The high biomass biofumigants, when chopped and incorporated, are then likely to produce ITC at sufficiently effective concentrations. The average maximum temperature for September and October 2018 was recorded as 17.6 °C and 14.5 °C whereas in the previous five-year period (2013–2017), the average maximum temperature in September and October were 18.2 °C and 15.1 °C, respectively. Moreover, the average minimum temperature in September 2018 was 8.8 °C and in October 2018 was 6.1 °C which were 0.6 and 1.6 degrees, respectively, lower than the average for the previous five years [45]. Overall, these two months, when the brassica crops were growing, were slightly cooler compared to the past years. Although the average maximum temperature for August 2018 (21.3 °C) was a degree higher than the average for the 2013–2017 period, the late sowing (13 August 2018) meant that most of the higher temperatures and longer daylight hours were missed. It would have been better if the cover crops were sown in the first week of August which would have probably resulted in higher biomass and higher GSL concentration in the brassica tissues. Moreover, the sun hours in November 2018 (50 h) were lower than the average sun hours for November (61 h) for 2013–2017 period [45]. The lower sun hours in the month, when brassica crops were to be incorporated, might have caused low GSL concentrations.Estimation of ITC-release potential based on GSL concentration and biomass [46] indicates that sinigrin in B. juncea shoots in the present study would produce AITC concentrations at 40 nmol g−1 soil, assuming a soil bulk density of 1.4 g cm−3 and incorporation to 20 cm. However, this concentration is estimated assuming complete conversion of sinigrin in above-ground tissue to AITC, whereas practically 1% ITC release-efficiency has been reported [46]. Hence, true AITC concentrations were likely to be even lower. In previous in vitro assays, AITC ED50 for F. graminearum was found to be 99 mg L−1 [27], which is equivalent to 998 nmol ml−1. This suggests that the potential AITC in B. juncea plots (≤40 nmol g−1 soil) were very low compared to effective AITC concentrations. Requirement of higher ITC concentrations has also been reported for other ITC such as, methyl ITC concentrations of 517 to 1294 nmol g−1 soil are estimated to be required for soil sterilisation [54]. Incorporation of B. oleracea var. caulorapa L. resulted in a mean F. graminearum DNA and DON in maize more than 50% lower compared to fallow. Although the effect was not significant, it does suggest a weak biofumigation effect may have occurred. Growth of B. oleracea var. caulorapa L. was patchy and lower (fresh wt. 20 t ha−1) than the other brassicas. A greater biomass of this brassica might have suppressed F. graminearum growth more efficiently and subsequently decreased DON concentrations in maize significantly. Fan et al. [55] demonstrated inhibitory effect of B. oleracea var. caulorapa L. against mycelial growth of F. graminearum under in vitro conditions. They reported that 10 g of powdered frozen tissue per Petri dish inhibited the mycelial growth by 70% on day 4 declining to 51% on day 7. In addition to its suppressive effect against fungi [55], B. oleracea var. caulorapa L. has been found effective in controlling nematodes too. Mashed leaves of B. oleracea var. caulorapa L. reduced population density of the nematode, Meloidogyne incognita (infecting cowpea plant) by 78–80%, whereas reduction by metam sodium was 43–65% in pot experiments [56]. Brassica oleracea var. caulorapa L. appears to be a potential biofumigant for suppressing F. graminearum in field, hence further investigation on the biofumigation effect of this brassica using different cultivars is recommended. Fusarium graminearum DNA content in maize was significantly reduced by R. sativus. This is in agreement with findings of a pot experiment where macerated tissue of R. sativus at a rate of 10 g kg−1 soil significantly reduced F. graminearum DNA content in soil [57]. In the same study, macerated tissue of R. sativus (5 g per Petri dish) also reduced mycelial growth of F. graminearum by 17–19%. The reduction in F. graminearum DNA content in maize obtained with R. sativus may also be attributed to “partial biofumigation”. The thick roots of R. sativus, giving a high under-ground biomass, might have released high concentrations of GSL that were probably hydrolysed by myrosinase activity of soil microorganisms [58,59], thus producing biofumigation effect. Ngala et al. [33,60] observed partial biofumigation under a growing R. sativus crop, causing suppression of the potato cyst nematode Globodera pallida in glasshouse and field conditions.

The present study failed to identify a significant suppression of F. graminearum inoculum in soil, and disease and DON contamination in maize, however some of the observed reductions in F. graminearum DNA and DON warrant further research. Future studies on optimising agronomy and increasing GSL concentration in brassicas are recommended to achieve potentially successful biofumigation effect. For example, approaches to maximise biomass production, such as, under sowing in the previous crop would allow more time for brassicas to grow and probably result in higher biomass and greater GSL concentration in the brassica tissues. Selection of brassica with the most suitable GSL profile and concentrations, and good biomass are important factors affecting the outcome of biofumigation. Therefore, to achieve a successful biofumigation effect, the approach needs to be optimised considering environmental factors, such as, temperature and moisture content of soil, as well as establishment and growth of the biofumigant crop.

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