Fusarium head blight in the Russian Far East: 140 years after description of the ‘drunken bread’ problem

Introduction

The first description of Fusarium head blight (FHB) within the territory of Russia was in the Far East in 1882 (Palchevsky, 1891; Voronin, 1890). This region is typically a monsoon climate with very damp and warm summers due to the influences of the Sea of Japan and the Pacific Ocean. The scientific investigation of this disease began at the end of the 19th century, but long before this, Chinese peasants and later Russian settlers related the poisoning of people and animals with pinkish grains and heads in the fields.

Between 1882 and 1914, epidemics of this disease in the Far East occurred almost every year (Naumov, 1916). Consumption of affected grain and straw caused numerous cases of food poisoning of people and farm animals. The initial signs and symptoms of the disease resemble those that can develop after drinking too much alcohol (including dizziness and headache, trembling hands, confusion, and vomiting) and thus the disease was named ‘drunken bread’. The extensive research undertaken by Russian mycologists revealed that Fusarium roseum Link (F. graminearum Schwabe) with teleomorph stage Gibberella saubinetii Sacc. (G. zeae [Schwein.] Petch) was the principal cause of the disease (Jaczewski, 1904; Naumov, 1916; Voronin, 1890).

Palchevsky (1891), who lived in this territory and was one of the first to report the disease of grain crops, studied its etiology and deposited diseased grain head specimens in herbaria (the first specimens, kept in the Herbarium LEP of our laboratory, are dated 1912). Thanks to this inquisitive individual, drawings of typical symptoms of the disease and pathogens were published (Fig. 1).

FHB was a persistent problem in the Far East during the 20th century (Abramov, 1938; Naumov, 1916) and continues to be today. High severities of FHB are reported nearly every year in the region. Mycological analyses of seed samples from 1998–2002 have shown a high level of FHB-infected wheat and barley seed (23–32%). The most frequently isolated pathogen was F. graminearum (Gagkaeva et al., 2002; Ivashchenko, Shipilova & Levitin, 2000).

Potential toxic effects of mycotoxins associated with FHB, particularly trichothecenes, which are secondary metabolites produced by F. graminearum and other Fusarium species, , can result in numerous health problems after consumption of infected grain, flour, and processed products. Fusarium outbreaks are a concern because of loss of grain yield and quality and mycotoxin contamination.

The development of multilocus sequence typing (MLST) has facilitated the identification of species and chemotypes of the F. graminearum species (Fg) group (Ward et al., 2008). Among them, the ubiquitous F. graminearum sensu lato (s. lat.) includes at least 16 phylogenetic species (Aoki et al., 2012; O’Donnell et al., 2000; O’Donnell et al., 2004; O’Donnell et al., 2008) united into the Fg group. Based on MLST assays, several species of the Fg group, including F. graminearum sensu stricto (s. str.), F. ussurianum T. Aoki, Gagkaeva, Yli-Mattila, Kistler & O’Donnell, and F. vorosii B. Tóth, Varga, Starkey, O’Donnell, H. Suga & T. Aoki, were identified in the grain grown in the Russian Far East (Yli-Mattila et al., 2009). A biogeographic hypothesis suggests that F. vorosii, F. ussurianum, and F. asiaticum O’Donnell, T. Aoki, Kistler & Geiser may be endemic Asian species within the Fg group (O’Donnell et al., 2004).

All species within the Fg group are capable of producing type B trichothecenes, but the activity of their formation is largely different. Three types of chemotypes have been identified among the strains: deoxynivalenol (DON) and 3-acetyldeoxynivalenol (3-AcDON), DON and 15-acetyldeoxynivalenol (15-AcDON), and nivalenol and 4-acetyl-nivalenol (NIV) (Moss & Thrane, 2004; Ward et al., 2002).

High humidity and heavy rainfall stimulate the development of F. graminearum s. lat. in grain and, as a result, increase its contamination by DON (Aldred & Magan, 2004; Ramirez, Chulze & Magan, 2006). The Russian Far East is often exposed to weather disasters, such as floods, which can lead to negative consequences insurmountable by human efforts and technologies resulting in significant agricultural damage. In the summer of 2019, in the Amur region, which accounts for 60–70% of grain production in the Russian Far East, flooding after substantial rainfall has wreaks havoc with extensive damaged crops. In this region, an emergency regime was established on July 25, 2019, and ‘about 250,000 ha was flooded, which amounted to about 20% of the total cultivated area in the region’ (TASS, 2019). As a result, the yield of cereals was only partially saved and harvested.

Despite the long history of the problem in the Far East, there is still no objective information on infection and mycotoxin contamination of harvested grain. Epidemics of FHB in the region fuelled our interest in investigating this disease using available modern methods of research.

Broad geographic surveys of Fusarium species on cereal crops are important to establish if any present shifts in populations occur in response to environmental change. We expected the diversity of Fusarium species belonging to the Fg group in this territory to be high, since the conditions were very favorable for fungi and led to the disease epidemic. In addition, we assumed that F. asiaticum may appear in the complex of pathogens, since in the neighbouring countries of China and Japan this species is detected on cereals with a high frequency (Gale et al., 2002; Láday et al., 2004; Qu et al., 2007; Suga et al., 2008). In China, where the problem of FHB is also acute, two species of the Fg group have been identified: F. graminearum s. str. and F. asiaticum (Qu et al., 2007). In the north of China, closest to the Amur Region, mostly F. graminearum was dominant, and all of strains were the 15-AcDON chemotypes (Shen et al., 2012). But F. asiaticum was the predominant species in the Yangtze River Basin, and chemotypes of strains were either 3-AcDON or NIV, with 3-AcDON being predominant.

This study aimed was to investigate (1) the natural Fusarium species occurrence and mycotoxin contamination of grain from the Amur region in the Far East in the most favorable conditions for pathogens and (2) provide the multilocus analysis of isolated strains of F. graminearum sensu lato to species and trichothecene genotype diversity.

Materials & Methods

Results

Detection of grain infection with fungi

Visual analysis of grain samples revealed the presence of various deformities, shrunken and with a pink-white coloration of grains in the amount of 5–42% (Fig. 2). Due to prolonged wet weather, the salmon-orange conidia masses of the fungus and blue-black perithecia can be seen on the infected spikelet and glumes in barley. Most of the perithecia were mature, and when placed in a water drop, the ascospores with three septa appeared from asci.

The average germination of wheat grain was 25.1% (12–41%) and of barley grain was 55.3% (48–62%). Almost 100% infection by fungi of all grain samples was noted; often, different fungi were isolated from one grain.

Mycology analyses verified that infection by Fusarium spp. was the primary cause of damage in grains, and infection rates reached extremely high percentages (Table 3). Moreover, the proportion of F. graminearum s. lat. strains among all isolated Fusarium spp. averaged 83.7% in the wheat grain and 89.7% in the barley grain. Fusarium sporotrichioides Sherb. strains were detected in 61% of samples, but grain infection was low (1–4%) (Table S1). Among the isolated fungi, the occurrence of F. avenaceum (Fr.) Sacc., F. anguioides Sherb., F. tricinctum (Corda) Sacc., F. poae (Peck) Wollenw., F. cerealis (Cooke) Sacc., F. equiseti (Corda) Sacc., F. incarnatum (Desm.) Sacc., and F. heterosporum Nees et T. Nees as well as four strains belonging to the Fusarium fujikuroi species complex was lower (Table S1).

Table 3:

Fungal and mycotoxin contamination of wheat and barley grain grown in the Amur region in the Russian Far East, 2019.

Parameters		Samples of grain
		Wheat	Barley
Grain infected with fungi on average (range), %	Fusarium spp.	81.1 (58–98)	80.5 (64–94)
	incl. F. graminearum	68.0 (47–88)	72.0 (61–92)
Content of mycotoxins on average (range), ppb	DON	7,498 (3,207–13,343)	5,390 (912–7,756)
	3-AcDON	122 (27–293)	131 (0–192)
	15-AcDON	85.5 (23–179)	93.5 (19–154)
	3-DON-glucoside	1,011 (299–2,001)	2,128 (98–3,803)
	ZEN	1,153 (92–3,670)	537 (111–928)
	MON	70.2 (10–218)	72.7 (5–207)
Amount of Fusarium DNA ×10−3 on average (range), pg/ng	F. graminearum	6,089 (2,658–11,342)	2,102 (163–3,557)
	3-AcDON genotype	1,084 (395–2,007)	508 (107–783)
	15-AcDON genotype	1,708 (755–2,776)	371 (101–713)
	F. avenaceum	40 (6–97)	13 (3–38)

DOI: 10.7717/peerj.12346/table-3

Alternaria spp. were the second frequent genera isolated from the grain samples. Moreover, the infection of wheat grain with Alternaria spp. was almost two times lower (12.9%) than that of barley grain (21.5%). Cladosporium spp., Clonostachys rosea (Link: Fr.) Schroers, Samuels, Seifert & W. Gams, Cochliobolus spp., Epicoccum nigrum Link, and other fungi were also identified in the grain mycobiota (the Table S1).

Genotyping of Fusarium spp.

Multilocus analysis of the TEF, URA, RED, and Tri101 sequences were used to determine the phylogenetic relationships among Fusarium strains. The dataset included 34 combined sequences of the analyzed strains as well as the 12 reference sequences of Fusarium spp. belonging to the Fg group and consisted of a total of 2,941 characters (612 bp from the TEF, 558 bp from URA, 821 bp from RED, and 950 bp from Tri101). The sequence of the F. pseudograminearum type strain NRRL 28334 was used as the outgroup. The resulting phylogenetic tree based on DNA sequence data of Fusarium species was constructed (Fig. 3). Maximum likelihood and maximum parsimony bootstrap support values greater than 70%, followed by Bayesian posterior probability scores greater than 0.95, are shown at the nodes.

The topology of phylogenetic trees constructed by different methods turned out to be similar and demonstrated the phylogenetic relationships between species established earlier (Aoki et al., 2012). Twenty-nine analyzed Fusarium strain isolated from Amur grain and one strain from Kemerovo region belonged to the clade with reference strain NRRL 5883 F. graminearum s. str. (Fig. 3). Among the analyzed strains of F. graminearum s. str., nine strains were the 3-AcDON chemotype while 21 strains turned out to be the 15-AcDON chemotype (Table 2).

From four doubtful Fusarium strains, one strain MFG 60604, isolated from wheat grain from the Altai Krai (West Siberia), was clustered with the reference strain F. vorosii NRRL 45790 with high bootstrap support (ML/MP/BP: 99/99/1.0). Our phylogenetic analysis indicates that strain MFG 60604 is F. vorosii and it is determined as a 15-AcDON chemotype.

Three other doubtful strains, MFG 58836, MFG 59052, and MFG 60755, formed the clade with the reference strains F. culmorum NRRL 25475 with high bootstrap support (ML/MP: 98/100). All three F. culmorum strains were the 3-AcDON chemotype (Table 2).

Discussion

Despite the long history of the FHB problem in the Russian Far East, objective data on pathogen composition and content of mycotoxins in naturally infected grain is clearly under-published. The mycological analyses of grain from this region in 2019 revealed extremely high infection of grain with Fusarium spp.—up to 98%. The predominant cause of FHB was the Fg group, which accounted for 86% of all isolated Fusarium spp.

Interestingly, the amount of fungal DNA in the wheat grain was on average higher than in the barley grain, while the percentage of infected grains was the same. The revealed differences may be due to the abundance of fungal biomass concentrated on the surface of barley grains (husk, palea, pericarp), while the wheat grain is completely permeated with fungal hyphae. In general, in this situation in 2019, the infection rates for both wheat and barley were off the scale. In our opinion, the highest DON content detected in this study, in the amount of 13,343 ppb, exceeds the maximum amounts of this mycotoxin in grain previously detected in the Russian territory. During the outbreak of FHB in South European part of Russia in 1985–1991 the maximal content of DON in grain reached 10,000 ppb (Kononenko, 2005). Recently, in 2017, DON amount of 7,920 ppb was detected in wheat grain grown in South European part of Russia (Kononenko, Burkin & Zotova, 2020).

The content of 3-AcDON in wheat and barley grain, as well as 15-AcDON, was similar and did not exceed 293 ppb. In the plant, DON can be present as a metabolite, DON-3gl, which is represents up to 46% of the total amount of DON in infected wheat and maize varieties (Berthiller et al. 2009). It has been shown that DON-3gl can be converted back to DON in mammals (Dall’Erta et al., 2013; Tucker et al., 2019). Therefore, DON-3gl is also frequently referred to as a masked mycotoxin. In our study, the maximum content of DON-3gl reached 3,803 ppb and was twice as high, on average, in barley grain than in wheat grain. The amounts of DON-3gl come to 13.5% and 39.5% of the total amounts of DON in infected wheat and barley samples, respectively. However, there were no significant differences in the content of the trichothecene mycotoxin average between wheat and barley grains.

Using morphology to accurately assess species limits for the Fg group is not reliable. Before this study, we hypothesized that in the extremely humid and warmest conditions of 2019, in the area where FHB outbreaks were observed for at least 140 years, several species of the Asian clade of the Fg group will be identified. Especially considering that earlier we have already found three species of the Fg group in this region: F. graminearum s. str., F. ussurianum and F. vorosii (Yli-Mattila et al., 2009). Selecting freshly isolated fungi for analysis, we took cultures for a detailed study, which included all the morphological diversity present within the limits possible for the Fg group (pigmentation, rate of formation of macroconidia, size, and shape). Multilocus phylogenetic analysis revealed that all strains from the Amur grains belonged to the F. graminearum s. str.

Molecular methods make it possible to reveal the intraspecific diversity of F. graminearum and to establish the quantitative presence of two different chemotypes. The F. graminearum strains are divided into 3-AcDON and 15-AcDON chemotypes depending on the prevailing acetylated form of DON (Alexander et al., 2011; Foroud et al., 2019). Regional differences have been reported regarding the occurrence of chemotypes within the Fg group (Foroud et al., 2019; Pasquali et al., 2016). In our study, on average, the DNA content of the 3-AcDON and 15-AcDON chemotypes of F. graminearum in the grain was similar, but the DNA of the 15-AcDON chemotype in wheat grain was significantly higher (4.6 times) than in barley (p = 0.014), whereas the difference in DNA content of the 3-AcDON fungus chemotype in wheat and barley grain was insignificant. It is not known whether the observed differences are related to chemotype-specific plant-host preferences. There may be a difference in pathogenicity between the 3- and 15-AcDON chemotypes to wheat and barley (Foroud et al., 2019; Clear et al., 2013).

According to our results, 30% of the analyzed F. graminearum strains were the 3-AcDON chemotype, while 70% of the strains were the 15-AcDON chemotype. Previously, the chemotype analysis of the 105 F. graminearum strains collected in the Russian Far East in 1998–2006 revealed the approximately equal occurrence of 3-AcDON (48%) and 15-AcDON (52%) chemotypes (Yli-Mattila et al., 2009). An increase in the 15-AcDON chemotype has recently been shown in regions of Europe, where the 3-AcDON chemotype was previously dominant, although many of the factors affecting their distribution are still unclear (Nielsen et al., 2012; Aamot et al., 2015; Pasquali et al., 2016; Foroud et al., 2019). The third chemotype of F. graminearum s. str. producing nivalenol (NIV) has not yet been identified in Russia or China (Shen et al., 2012), although it is known to be found in Europe (Pasquali et al., 2016).

In our analysis, Fusarium sp. strain MFG 60604 was included that was isolated from wheat grain in the West Siberian region (the Altai Krai); phenotypically, this strain was a dubious representative of the Fg group. In this region, the occurrence of F. graminearum was previously not typical, but in recent years, we have been identifying this pathogen in cereal grains (Gagkaeva et al., 2019). The strain MFG 60604, isolated from wheat grain from the West Siberia, was clustered with the reference strain F. vorosii NRRL 45790 with high bootstrap support (ML/MP/BP: 98/99/1.0), which allows for accurate establishment of its species affiliation. A single strain (MFG 60604) identified as F. vorosii in this study, is the only third strain of F. vorosii found in Russia and the first one identified in the Siberian region. Previously identified strains of F. vorosii from the Russian Far East belonged to the 15-AcDON chemotype (Yli-Mattila et al., 2009) and so did the strain identified in this study. However, among six F. vorosii strains originating from Korea, five were the NIV chemotype, while only one was the 15-AcDON (Lee et al., 2016). Among F. vorosii, no strains of the 3-AcDON chemotype have been identified, which, probably, were not detected due to the small number of strains of this species analyzed at present. In the limited surveys at present, strains of several species of the Fg group were found to represent only a single chemotype (Aoki et al., 2012).

Two strains of F. culmorum of closely related taxon to Fg group from the West Siberian and Ural regions and one from the South European region of Russia were included in the study. The high genetic similarity of analyzed F. culmorum strains collected from remote regions characterized by different climatic conditions (the distance between isolation points is about 2,500 km) is consistent with the previously shown information that F. culmorum is a single phylogenetic species with little or no differences between lineages, despite the geographic separation of genotypes (Obanor et al., 2010).

The studies analyzing the occurrence of F. culmorum chemotypes in different regions, as a rule, show a significant excess of the occurrence of the DON chemotype compared to the NIV chemotype (Laraba et al., 2017; Pasquali et al., 2016; Scherm et al., 2012). Strains of the 15-AcDON chemotype typical for F. graminearum were not identified among the strains of F. culmorum. A previous analysis of a few strains of F. culmorum from the Russian territory has also characterized them as the 3-AcDON chemotype (Yli-Mattila et al., 2009).

Fusarium spp. continue to pose a threat to farmers, destroying crops or dramatically reducing yields, as well as to animal and human health due to the production of mycotoxins. Even in our time, when we know much more about the nature of Fusarium spp. then 140 years ago, we are still not very successful in controlling the diseases they cause on crops if conditions are favorable for the development of pathogens. Indeed, in the process of our study, it was shown in the mass media that although the grain was harvested with great difficulty, due to the significant contamination of the grain, part of the crop, 240 tons, had to be destroyed by fire.

Conclusions

The high prevalence of Fusarium head blight in cereal grains cultivated in the Far East is particularly alarming and strongly indicates the need for increased measures to prevent plant infection and improved food safety interventions. The maximum DON content in wheat grains reached 13,141 ppb in this study. The multilocus sequence revealed that the majority of the strains used in this study belonged to F. graminearum s. str.

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