At the scene of the crime: New insights into the role of weakly pathogenic members of the fusarium head blight disease complex

Abstract

Plant diseases are often caused by a consortium of pathogens competing with one another to gain a foothold in the infection niche. Nevertheless, studies are often limited to a single pathogen on its host. In Europe, fusarium head blight (FHB) of wheat is caused by multiple Fusarium species, including Fusarium graminearum and F. poae. Here, we combined a time series of (co)inoculations, monitored by multispectral imaging, transcriptional, and mycotoxin analyses, to study the temporal interaction between both species and wheat. Our results showed coinoculation of F. graminearum and F. poae inhibited symptom development but did not alter mycotoxin accumulation compared to a single inoculation with F. graminearum. In contrast, preinoculation of F. poae reduced both FHB symptoms and mycotoxin levels compared to a single F. graminearum infection. Interestingly, F. poae exhibited increased growth in dual infections, demonstrating that this weak pathogen takes advantage of its co‐occurrence with F. graminearum. Quantitative reverse transcription PCR revealed that F. poae induces LOX and ICS gene expression in wheat. We hypothesize that the early induction of salicylic and jasmonic acid‐related defences by F. poae hampers a subsequent F. graminearum infection. This study is the first to report on the defence mechanisms of the plant involved in a tripartite interaction between two species of a disease complex and their host.

Timing of Fusarium poae infection on wheat determines fusarium head blight development and mycotoxin production through a plant‐mediated defence mechanism.

1. INTRODUCTION

Plant diseases have been studied for decades as the interaction of a single pathogen with its host. However, it is now being recognized that plant diseases are often the result of multispecies interactions, including two or more pathogens from the same or even different kingdoms (Lamichhane and Venturi, 2015). Fusarium head blight (FHB) is one of the most important fungal diseases on wheat, causing yield losses of up to 40% (Parry et al., 1995). Although the hemibiotrophic fungus Fusarium graminearum sensu stricto (O’Donnell et al., 2004) is considered to be the primary causal agent of the disease in central Europe, North America, and Asia (Leplat et al., 2013; Backhouse, 2014), several species involved in FHB are frequently reported. In Europe, besides F. graminearum, F. culmorum, F. poae, F. avenaceum, Microdochium nivale, and M. majus are the predominant species (Ioos et al., 2005; Xu et al., 2005). Despite their presence in association with diseased kernels, only F. graminearum and F. culmorum are considered highly pathogenic. The other species are less aggressive on wheat in that they may infect but do not proliferate, grow superficially, or only thrive at the sites of inoculation (Stack and McMullen, 1985; Brennan et al., 2003).

In the interaction with wheat, F. graminearum first grows on the exterior parts of florets and glumes or between the cuticule and the epidermal cell wall (Bushnell et al., 2003). Finally, plant tissues are penetrated and the fungus spreads throughout the ear via the vascular system in the rachilla and rachis (Brown et al., 2010). This spread is facilitated via the production of the mycotoxin deoxynivalenol (DON) and its acetylated derivatives 3‐acetyl DON and 15‐acetyl DON, an important virulence factor causing necrosis of the ear tissue (Audenaert et al., 2014). During infection, F. graminearum corrupts the plant’s defence system by reducing the expression of defence genes and boosts its DON production in response to the plant's oxidative burst, which is a typical hallmark of first‐line plant defence (Desmond et al., 2008; Audenaert et al., 2014). A successful plant defence against F. graminearum is meticulously regulated by a sequential defence signalling comprising salicylic acid (SA) and jasmonic acid (JA) during the early and later stages of infection, respectively (Ameye et al., 2015).

An important but more enigmatic member of the FHB complex is F. poae. Compared to other Fusarium members of the FHB disease complex, F. poae causes the least reduction of seed germination, hence F. poae can be considered a weak pathogen compared to other Fusarium species (Browne and Cooke, 2005). Despite its low virulence, F. poae is omnipresent in Europe (Xu et al., 2005; Audenaert et al., 2009; Vogelgsang et al., 2019), which is partially attributed to its higher resistance to fungicides compared to other Fusarium species. Treating wheat fields with azole fungicides causes the Fusarium population to shift from a population dominated by F. culmorum and F. graminearum to a F. poae‐dominated population (Audenaert et al., 2011). F. poae produces a complex blend of both type A (diacetoxyscirpenol [DAS] and neosolaniol [NEO]) and type B (nivalenol [NIV] and fusarenon‐x [FUS‐X]) trichothecenes whose function remains enigmatic (Sugiura et al., 1993; Thrane et al., 2004; Vogelgsang et al., 2008; Vanheule et al., 2017). However, no F. poae isolate has been identified that produces the virulence factor DON or its acetylated derivatives 3‐acetyl DON and 15‐acetyl DON, which are produced by the more aggressive pathogens in the FHB complex such as F. graminearum, F. culmorum, or F. asiaticum.

In this study, we hypothesized that F. poae depends on the presence of F. graminearum to infect wheat and that the presence of F. poae influences the disease symptom development of F. graminearum in a time‐dependent manner. However, using an approach of time series coinoculations combined with phenomics and transcript analyses, we could demonstrate that the interaction between both species of the FHB disease complex is far more complex, with highly divergent symptoms depending on the timing of inoculation. Our results show that F. poae primes plant defence, which hampers subsequent infection by F. graminearum. Moreover, F. poae takes advantage of its co‐occurrence with the highly virulent F. graminearum. Our study demonstrates that F. poae is a freerider rather than an innocent bystander at the FHB infection site.

2. RESULTS

2.1. In the field, F. poae and F. graminearum co‐occur in the same spikelets

By analysing the data of a 17‐year survey in seven locations scattered throughout Flanders, Belgium, we assessed the co‐occurrence of F. poae and F. graminearum. In total, more than 7,000 wheat ears and more than 40 cultivars were assessed for the presence of FHB members. This analysis showed that in the ears with symptoms F. poae occurred in 30.0% of the cases alone, in 31.2% F. poae co‐occurred with F. graminearum, and in 15.4% F. poae co‐occurred with F. graminearum and F. culmorum (Figure 1a,b). This multiyear, multilocation analysis showed a significant co‐occurrence of F. poae with F. graminearum and F. avenaceum under field conditions (Figure 1c). Because of the low incidence of F. avenaceum, we did not further focus on the interaction with this species.

FIGURE 1.

Presence of Fusarium poae alone or in combination with other Fusarium species on the same spikelets in field samples. (a) Prevalence of different Fusarium species in a large‐scale investigation from 2002 until 2019. For 2006, no ears with symptoms were found during the period of sampling, hence no data are present. (b) Species association with F. poae. Fp, percentage of samples only containing F. poae and no other Fusarium species; Fp × Fg, percentage of samples containing both F. poae and Fusarium graminearum (and possibly also Fusarium avenaceum and/or Microdochium nivale); Fp × Fc, percentage of samples containing both F. poae and Fusarium culmorum (and possibly also F. avenaceum and/or M. nivale), Fp × Fg ×Fc, percentage of samples containing F. poae, F. culmorum, and F. graminearum (and possibly also F. avenaceum and/or M. nivale); Fp × …, percentage of samples containing F. poae, F. avenaceum, and/or M. nivale (but no F. graminearum or F. culmorum). (c) Pairwise species co‐occurrence patterns between F. poae and F. avenaceum, and F. culmorum and F. graminearum. exp_cooccur, expected number of sites having both species; P (lower frequency), probability that the two species would co‐occur at a frequency less than the observed number of co‐occurrence sites if the two species were distributed randomly (independently) of one another; P (higher frequency), probability of co‐occurrence at a frequency greater than the observed frequency (Griffith et al., 2016)

2.2. Impact of F. poae on F. graminearum disease progression on detached wheat leaves

To investigate the co‐occurrence of F. poae and F. graminearum in the field, we used a previously established model system of detached wheat leaves (Ameye et al., 2015) to assess symptom development of green fluorescent protein (GFP)‐tagged F. graminearum PH‐1 and wild‐type F. poae 2516. To assess whether preinoculation of F. poae affected the outcome of the F. graminearum infection, we inoculated a F. poae conidial suspension one day before the F. graminearum inoculation. Remarkably, the preinoculation with F. poae resulted in complete abolition of F. graminearum disease symptom development and the phenotype of no necrotic lesions on the leaf resembled that of a singular F. poae infection. This phenotype of no necrotic lesions was confirmed by the efficiency of photosystem II F_V/F_M values that reflect the healthy condition of leaf. The F_V/F_M values of preinoculation with F. poae were significantly higher than in the single F. graminearum inoculation (Figures 2a,b and S1). In addition, the lack of symptoms coincided with a decreased corrected (c)GFP signal, reflecting reduced presence of F. graminearum (Figure 2a,c) and a significant decrease in F. graminearum biomass (Figures 2d, S1, and S2). Finally, the typical reddish colour attributed to the presence of rubrofusarin, which is produced during an infection by F. graminearum, also disappeared when detached leaves were preinoculated with F. poae (Figure S3).

FIGURE 2.

Impact of Fusarium poae 2,516 inoculation on Fusarium graminearum PH‐1 infection in a detached leaf assay. (a) Pictures taken at three time points after infection (1, 2, and 3 days after inoculation [dai]). p(−2d) + g, preinoculation with F. poae 2,516 2 days before F. graminearum PH‐1 inoculation; w(−2d) + g, preinoculation with water 2 days before F. graminearum PH‐1 inoculation; p(−1d) + g, preinoculation with F. poae 2,516 1 day before F. graminearum PH‐1 inoculation; w(−1d) + g, preinoculation with water 1 day before F. graminearum PH‐1 inoculation; p + g, inoculation with F. poae 2,516 and F. graminearum PH‐1 at the same time; w + g, inoculation with water and F. graminearum PH‐1 at the same time; w + p, inoculation with water and F. poae 2,516 at the same time. (b) F_V/F_M (efficiency of photosystem II) values from 1 to 3 dai, n = 8. (c) Corrected green fluorescent protein (GFP) value from the GFP expressing PH‐1 strain signal from 1 to 3 dai, n = 8. (d) Normalized quantitative relative values (NRQ) of F. graminearum PH‐1 biomass, n = 4. Boxplots indicate the median (horizontal lines), 25th and 75th percentile range (boxes), and up to 1.5× interquartile range (whiskers). Different letters indicate significant differences between treatments (p < .05) for each time point

In the singular F. graminearum (w + g) inoculation, water‐soaked lesions developed and extended from day 2 onwards, which resulted in a decrease of the maximum F_V/F_M compared to the mock‐inoculated control leaves (Figures 2a,b and S4). In the singular F. poae (w + p) infection, no symptoms developed and F_V/F_M values of the detached leaves inoculated with F. poae were not significantly different from the mock‐inoculated with water leaves (Figure S4), which demonstrates that F. poae cannot cause symptoms on its own (Figure 2a,b). In the dual inoculations in which F. poae and F. graminearum were inoculated at the same time, symptoms were significantly less severe compared to the single F. graminearum infection. These results were confirmed by assessing the presence and activity of F. graminearum visualized by the cGFP signal and its biomass assessed by quantitative reverse transcription PCR (RT‐qPCR). Both parameters showed significant and a slight reduction respectively at day 3 compared to a singular infection with F. graminearum (Figure 2c,d). To assess whether there is direct antagonism between F. poae and F. graminearum in vitro, we coinoculated both species on potato dextrose agar (PDA) plates and no obvious mutual antagonism was observed (Figure S5).

To understand the impact of F. graminearum PH‐1 on the presence of F. poae 2,516 we used a GFP‐tagged F. poae 2,516 isolate in combination with a wild‐type F. graminearum PH‐1 and assessed the impact of the latter on F. poae presence. These experiments showed that a singular F. poae infection resulted in less F. poae biomass at day 3 (Figure 3a–c) and a reduced cGFP signal at day 4 compared to the leaves in which F. poae was inoculated with F. graminearum at same time (Figure S6). We inoculated the GFP‐tagged F. poae on leaves which were mechanically wounded by scratching the epidermal layer over a length of 0.1, 1 and 2 cm with a sterile scalpel to check whether F. poae benefits from the leaf damage induced by F. graminearum infection. F. poae showed a significantly larger growth in larger wounds (Figure S7).

FIGURE 3.

Impact of Fusarium graminearum PH‐1 inoculation on Fusarium poae 2,516 infection in a detached leaf assay. (a) Green fluorescent protein‐tagged F. poae 2,516 inoculation from 1 to 3 days after inoculation (dai). p(−1d) + g, preinoculation with F. poae 2,516 1 day before F. graminearum PH‐1 inoculation; p + g, inoculation with F. poae 2,516 and F. graminearum PH‐1 at the same time; w + g, inoculation of water and F. graminearum PH‐1 at the same time; w + p, inoculation with water and F. poae 2,516 at the same time. (b) Corrected green fluorescent protein (GFP) value signal from 1 to 3 dai, n = 8. (c) Normalized quantitative relative values (NRQ) of F. poae 2,516 biomass on leaves, n = 4. Boxplots indicate the median (horizontal lines), 25th and 75th percentile range (boxes), and up to 1.5× interquartile range (whiskers). Different letters indicate significant differences between treatments (p < .05) for each time point

Finally, we monitored disease progress of F. graminearum by inoculating F. poae 1 or 2 days after F. graminearum. The F. poae inoculation did not influence the symptom development of F. graminearum in this experiment (Figure S8).

To verify our observations in wheat ears, the main niche of FHB pathogens, we assessed whether preinoculation of F. poae also reduced the infection of F. graminearum on ears. We therefore inoculated a F. poae conidial suspension 1 or 2 days before inoculating the same spikelets with F. graminearum. After 7 days of infection, preinoculation of spikelets with F. poae 1 or 2 days before F. graminearum resulted in a not statistically significant trend of a decrease of FHB symptoms in ears, as measured by red‐green‐blue (RGB) images, higher F_V/F_M values (Figures 4a,b and S9), the percentage of low scored symptom ears (Figure 4d), a reduced cGFP signal of F. graminearum (Figures 4a,c and S9), and a reduction in F. graminearum biomass by RT‐qPCR (Figures 4e and S9) compared to singular inoculation of F. graminearum or coinoculation of F. graminearum and F. poae, confirming our findings from the detached leaf assays.

FIGURE 4.

Impact of Fusarium poae 2,516 inoculation on Fusarium graminearum PH‐1 infection on wheat ears. (a) Representative pictures taken at 7 days after inoculation (dai). p(−2d) + g, preinoculation with F. poae 2,516 2 days before F. graminearum PH‐1 inoculation; w(−2d) + g, preinoculation with water 2 days before F. graminearum PH‐1 inoculation; p(−1d) + g, preinoculation of F. poae 2,516 1 day before F. graminearum PH‐1 inoculation; w(−1d) + g, preinoculation of water 1 day before F. graminearum PH‐1 inoculation; p + g, inoculation with F. poae 2,516 and F. graminearum PH‐1 at the same time; w + g, inoculation with water and F. graminearum PH‐1 at the same time; w + p, inoculation with water and F. poae 2,516 at the same time. (b) F_V/F_M (efficiency of photosystem II) values at 7 dai, n = 8. (c) Corrected green fluorescent protein (GFP) value from the GFP expressing F. poae 2,516 mutant signal at 7 dai, n = 8. (d) Infection level after 7 dai. (e) Normalized quantitative relative values (NRQ) of F. graminearum PH‐1 biomass at 1, 2, 4, and 7 dai, n = 4. Boxplots indicate the median (horizontal lines), 25th and 75th percentile range (boxes), and up to 1.5× interquartile range (whiskers). Different letters indicate significant differences between treatments (p < .05) for each time point

2.3. Gene expression analysis of trichothecene production

We analysed transcripts of the TRI5 gene in F. graminearum, a key gene in DON biosynthesis. Both in the wheat leaf assay and in wheat ear assay, preinoculation with F. poae 1 or 2 days before inoculation with F. graminearum resulted in a significantly lower level of TRI5 gene expression compared to a singular F. graminearum inoculation at early infection time points (days 1 and 2; Figures 5a,c and S10). At later time points (day 3 for leaves assay, days 4 and 7 for ears assay), the differences of TRI5 gene relative expression between treatments was less pronounced except for the interaction in which F. poae was inoculated 2 days before an inoculation with F. graminearum and the interaction in which both of the species were inoculated at the same time in the leaf assay. For the TRI5 gene expression in F. poae, the biomass of F. poae is very low and the expression of TRI5 was below the detection level.

FIGURE 5.

Impact of Fusarium poae 2,516 on mycotoxins produced by Fusarium graminearum PH‐1. (a) Expression profile of the TRI5 gene in F. graminearum PH‐1 during a period of 3 days on wheat leaves. Data represent four biological replicates. The expression of the TRI5 gene from F. poae is below detection level as the 2,516 biomass is very low. (b) Mycotoxin concentrations 4 days after inoculation of wheat leaves with F. graminearum PH‐1. Data represent four biological replicates. (c) Expression profile of the TRI5 gene in F. graminearum PH‐1 during a period of 7 days on wheat ears. Data represent four biological replicates. The expression of TRI5 gene in F. poae is below detection level as the biomass is very low. (d) Mycotoxin concentrations 7 days after inoculation on wheat ears with F. graminearum PH‐1. Data represent five biological replicates. Boxplots indicate the median (horizontal lines), 25th and 75th percentile range (boxes), and up to 1.5× interquartile range (whiskers). Different letters indicate significant differences between treatments (p < .05) for each time point

2.4. Effect of F. poae on the production of type B trichothecenes during F. graminearum infection in detached leaves and ears

To assess the impact of F. poae on the presence of the type B trichothecenes produced by F. graminearum (DON, 15‐ADON, and 3‐ADON), these mycotoxins were quantitatively determined in leaves and ears at the end of the experiment at 4 and 7 days, respectively, using liquid chromatography coupled to mass spectrometry (LC‐MS/MS) (Figures 5 and S10). In detached leaves, the singular F. graminearum infection resulted in the highest DON level, with a mean concentration of 69 mg/kg, followed by the coinoculation treatment of F. poae and F. graminearum at 45 mg/kg. All these treatments resulted in median DON concentrations of more than 20 mg/kg of leaf material. Preinoculation of leaves with F. poae 1 day before F. graminearum resulted in a significantly lower concentration of DON (3.1 mg/kg) compared to singular F. graminearum inoculations (Student's t test, p = .011). Also, for the DON metabolite 15‐ADON, lower concentrations were found if leaves were inoculated with F. poae 1 day before F. graminearum (1.2 mg/kg) compared to singular F. graminearum inoculations (2.4 mg/kg). For 3‐ADON concentrations were relatively low 0.6 mg/kg compared to 1.0 mg/kg, and were similar in all treatments irrespective of the coinoculation with F. poae. As PH‐1 is a 15‐ADON chemotype (Alexander et al., 2011), thus not a 3‐ADON producer, these trace levels may be attributed to spontaneous chemical conversion of 15‐ADON to 3‐ADON. DON‐3G is generated by plants via a detoxifying mechanism, where a glucose moiety is added to DON. The highest DON‐3G concentration was observed in interactions were F. graminearum was inoculated alone or in simultaneous coinoculations with F. poae (Figures 5b and S10).

A similar approach was pursued in wheat ears. All DON concentrations of wheat ears inoculated by F. graminearum or simultaneously inoculated with F. poae and F. graminearum resulted in similar DON levels of around 12 mg/kg. Preinoculation of ears with F. poae 1 day before F. graminearum resulted in significant reduction of the DON concentration (5.5 mg/kg) compared to singular F. graminearum inoculations (12.8 mg/kg, Student's t test, p = .01). Also, for the DON metabolite 15‐ADON lower concentrations were found if ears were inoculated with F. poae 1 day before F. graminearum (0.4 mg/kg) compared to singular F. graminearum inoculations (0.6 mg/kg). For 3‐ADON concentrations were relatively low, 0.07 mg/kg compared to 0.14 mg/kg, and similar in all treatments irrespective of the coinoculation with F. poae. The highest DON‐3G concentration was observed in interactions where F. graminearum was inoculated alone or in simultaneous coinoculations with F. poae (Figures 5d and S10).

We quantified the presence of NIV, FUS‐X, NEO, and DAS in both detached leaves and ears. Surprisingly, none of these trichothecenes were detected in any of the samples, indicating levels below the limit of detection (NIV 30 µg/kg, FUS‐X 31 µg/kg, NEO 39 µg/kg, and DAS 41 µg/kg).

2.5. Effect of F. poae and F. graminearum infection on SA and JA responses, and PR‐gene expression in detached wheat leaves and ears

To study the impact of a singular F. graminearum or F. poae infection on the SA and JA responses in detached leaves and ears, we analysed the expression level of genes encoding hallmark enzymes phenylalanine ammonia‐lyase (PAL) and isochorismate synthase (ICS) for the SA biosynthesis, and lipoxygenases LOX1 and LOX2 for the biosynthetic pathway leading to JA.

In the detached leaf assay, F. graminearum infection (w[−2d] + g, w[−1d] + g, and w + g) resulted in a trend of PAL up‐regulation (Figure S11a) and a down‐regulation of ICS (Figure S11b) at all measured time points. For LOX2, a down‐regulation was observed at days 2 and 3 in w + g, and at days 1 and 3 in w(−1d) + g (Figure S11d), while the expression of LOX1 was up‐regulated in w(−1d) + g and w + g at day 1, and w(−2d) + g and w(−1d) + g at day 3 compared to the control (w[−2d] + w, w[−1d] + w, and w + w; Figure S11c).

Infection with F. poae (w + p) resulted in a completely different profile of the SA and JA primary biosynthesis genes. An early induction of both LOX1 and LOX2 trends was observed at 1 day after inoculation (Figure S11c,d). In addition, a transient induction of ICS was observed at 2 days after inoculation (Figure S11b). These results show that although F. poae does not cause any symptoms, it does induce defence responses in detached leaves.

In addition, we also assessed the expression profiles of three important pathogenesis‐related (PR) genes (PR1, PR4, and PR5) as well as peroxidase (PEROX). Despite the fact that F. poae did not cause symptoms in detached leaves, a clear and consistent induction of all three PR genes was observed at all time points (Figure S11f–h). PEROX, which constitutes an early response to pathogens, showed a transient induction in response to F. poae alone (Figure S11e). These genes were also induced by F. graminearum, although the expression levels were higher in the latter interaction.

In wheat ears, the infection of F. graminearum (w[−2d] + g, w[−1d] + g, and w + g) resulted in a trend of consistently higher induction of PAL in the first 7 days (Figure 12a) while ICS was suppressed at all time points (Figure S12b). For LOX1 and LOX2, no clear effects were observed at days 1 and 2 but a suppression of LOX2 was observed at days 4 and 7 (Figure S12c,d). This response was highly similar to the F. graminearum response in the detached leaf assay, which shows that the detached leaf assay is an adequate high‐throughput alternative for wheat ear infections (Figure S11c,d). The infection with F. poae (w + p) resulted in an up‐regulation of PAL from day 1 onwards except for day 2 (Figure S12a). In addition, LOX1 (Figure S12c) and ICS (Figure S12b) were induced at days 2 and 7, respectively. Similar to the response in the detached leaf assay, the expression profiles of three important PR genes (PR1, PR4, and PR5) and PEROX showed a clear and consistent induction at all time points in both F. poae and F. graminearum (Figure S12e–h).

2.6. Defence response after dual infection of detached wheat leaves and wheat ears with F. poae and F. graminearum

When F. poae was inoculated on detached wheat leaves 1 or 2 days before the F. graminearum infection (p[−2d] + g and p[−1d] + g), no suppression of ICS or LOX2, which was characteristic of an inoculation with F. graminearum alone (w[−2d] + g and w[−1d] + g), was observed. On the contrary, in the treatments in which F. poae was preinoculated 1 or 2 days before F. graminearum (p[−2d] + g and p[−1d] + g), a slight induction of PAL and ICS was observed 2 days after the F. graminearum infection (which was 3 days after the F. poae infection; Figure S11). Finally, when we preinoculated F. graminearum before F. poae (g[−1d] + p), expression of the defence genes PAL, PR1, PR4, PR5, and PEROX was higher than the singular F. poae infection, while ICS was relatively lower (Figure S13).

The kinetics of the gene expression were more complex in wheat ears compared to wheat leaves. First of all, the singular inoculation with F. graminearum or the simultaneous inoculation of F. graminearum at the same time as F. poae resulted in a reduction in the expression of ICS at days 4 and 7. This reduction was less pronounced when F. poae was preinoculated 1 day before F. graminearum. In regard to expression of LOX1, the induction that was observed in detached wheat leaves upon F. poae inoculation was confirmed in wheat ears: singular F. poae inoculation and F. poae inoculation 1 day before F. graminearum inoculation resulted in an up‐regulation of LOX1 while singular F. graminearum inoculation resulted in a down‐regulation of LOX1. Similar to detached wheat leaves, all treatments except for the control treatments resulted in an induction of PR1, PR4, PR5, and PEROX (Figure S9e–h). The induction of these genes, especially of PEROX, was higher in interactions in which F. graminearum was inoculated alone compared to interactions in which F. poae was inoculated alone or in which F. poae was inoculated 1 day before F. graminearum.

2.7. Principal component analysis of gene expression, fungal biomass cGFP and NRQ, and the health condition of the plant (F_V/F_M)

To study whether enhanced defence against F. graminearum might play a role after preinoculation with F. poae, we performed a principal component analysis (PCA) on defence gene expression data, combined with multispectral data accounting for the fungal biomass (cGFP and normalized quantitative relative values [NRQ]) and the health condition of the plant (F_V/F_M) in both the leaf and ear assays.

In detached leaves at day 1 the control treatments (w[−2d] + w, w[−1d] + w, or w + w) clustered, were clearly distinct from all other treatments, and were separated along the first principal component. Analysis of the factor loadings (component coefficients) showed that the singular F. poae infection positively associated with the expression of LOX1 (lipoxygenase1) and LOX2 (lipoxygenase2). As the disease progressed, at days 2 and 3, several patterns emerged. The singular F. graminearum inoculations or the treatments in which F. graminearum was simultaneously applied with F. poae could be differentiated along the first principal component from all other treatments. The singular F. poae inoculations and the inoculations in which F. poae was applied 1 or 2 days before F. graminearum were more closely related to the control treatment. Analysis of the loading factors shows that ICS and LOX2 were positively correlated with F_V/F_M and contributed to the separation along the first principal component. Furthermore, the amount of fungal biomass as indicated by the cGFP signal and NRQ were negatively correlated with F_V/F_M, ICS, and LOX2 (Figures 6 and S14, and Table S1).

FIGURE 6.

Principal component analysis of gene expression, fungal biomass corrected green fluorescent protein (GFP) value from the GFP‐expressing PH‐1 mutant, and normalized quantitative relative values, and the health condition of the plant (F_V/F_M [efficiency of photosystem II]) from 1 to 3 days after inoculation (dai) and all time points in leaf assay. Data show the control treatments cluster separately from the w + g, p + g, w(−1d) + g, and w(−2d) + g treatments along the first principal component. The w + p, p(−1d) + g, and p(−2d) + g treatments are more closely related to the control treatment. Analysis of the factor loadings shows that F_V/F_M, ICS, and LOX2 are positively correlated and contribute to the separation along the first principal component. The size of the circles is proportional to their cos2 value

In ears, similar to leaf assay, from day 1 the control treatments clustered and were clearly distinct from all other treatments, and were split along the first principal component axis. Analysis of the factor loadings showed that the infection positively correlated with the expression of LOX1 and ICS and negatively correlated with the expression of LOX2 and PAL at day 1. At days 2, 4, and 7 control treatments, singular F. poae inoculations and inoculations in which F. poae was applied 1 or 2 days before F. graminearum infections could be separated from the other treatments along the first principal component. Analogously as in the leaf assay, a positive correlation was observed in these treatments with LOX1 or LOX2 and ICS gene expression (Figures 7 and S15, and Table S1).

FIGURE 7.

Principal component analysis of gene expression, fungal biomass corrected green fluorescent protein value, and normalized quantitative relative values, and the health condition of the plant (F_V/F_M [efficiency of photosystem II]) at 1, 2, 4, and 7 days after inoculation (dai) in ear assay. Data show the control treatments cluster separately from the w + g, p + g, w(−1d) + g, and w(−2d) + g treatments along the first principal component. The w + p and p(−1d) + g treatments are more closely related to the control treatment. A positive correlation was observed in these treatments with LOX1 or LOX2 and ICS gene expression. The size of the circles is proportional to their cos2 value

3. DISCUSSION

In plant pathology, the interaction of a plant with an invading pathogen is often considered as a bidirectional interaction involving a single pathogenic strain and its host. The outcome of this interaction is then typically determined by the mutual interplay of plant‐ and pathogen‐derived signals and pathways.

Yet, in some of the world's most important fungal plant diseases, several species or genera have been isolated from the tissue with symptoms, which suggests that multiple species and/or genera are involved in the cause and/or development of the disease (Lamichhane and Venturi, 2015). FHB in wheat can be caused by a complex of Fusarium species, black sigatoka disease in banana is caused by several Mycosphaerella species (Arzanlou et al., 2007), leaf spot on Eucalyptus is caused by several Teratosphaeria species (Crous et al., 2009), and potato leaf spot is caused by a complex of Alternaria species (Vandecasteele et al., 2018). Sometimes even different fungal genera are involved in one disease, for example grapevine decline caused by Botryosphaeriaceae spp. and Ilionectriaceae spp. (Whitelaw‐Weckert et al., 2013) and rice sheath rot disease caused by Fusarium spp. and Sarocladium oryzae (Bigirimana et al., 2015). Aphanomyces euteiches is an important root rot pathogen in pea. Despite the fact that Fusarium  solani does not cause symptoms in pea, coinoculation of plants with A. euteiches and F. solani resulted in significantly greater disease severity than A. euteiches alone, which points to a synergistic action of both fungi in the interaction with the plant (Peters and Grau, 2002).

Sometimes, even interkingdom associations are formed. As such, infection of maize by F. verticillioides is facilitated by the European corn borer (Ostrinia nubilalis; Blandino et al., 2015). The former synergistic action of an insect and a fungus causing a disease is not due to the insect vectoring the fungus. On the contrary, the European corn borer damages the maize plant and these wounds are used by F. verticillioides as entry points. Studies focused on these types of fungal diseases are still in their infancy and the available papers are often limited to the interaction of one member of the disease complex (often the most virulent one or the most frequently encountered one) with the host or focus on the identification of new species or genera in a disease complex (Lamichhane and Venturi, 2015, and the references therein).

Within the many different species involved in FHB, we aspired to unravel in detail the interaction between two predominant FHB species of wheat in Europe: the virulent pathogen F. graminearum, which often co‐occurs with the weak pathogen F. poae. Their co‐occurrence in several countries throughout Europe is counterintuitive as they behave quite differently on wheat ears. F. graminearum infects directly or infects through natural openings, after which it grows under the cuticle along stomatal rows (Pritsch et al., 2000). Subsequently, it colonizes glumal parenchyma cells. When it finally migrates to the rachilla and rachis, it starts to produce the necrotizing molecule DON (Audenaert et al., 2014). F. poae, on the other hand, is a weak pathogen that causes minor symptoms and infection is limited to the glumae (Siou et al., 2015). Despite their different lifestyles, they share the same host and ecological niche at the onset of infection, which leads to the assumption that they may interact with each other.

Using two model infection systems on detached leaves as well as on ears of intact plants, we observed that coinoculation of F. graminearum with the weak pathogen F. poae did not differ significantly from a singular F. graminearum infection. However, when detached leaves or ears were preinoculated with F. poae 1 day before F. graminearum infection, a clear reduction in disease symptoms was observed. In addition, we looked at the active fungal biomass by performing RT‐qPCR analyses and monitoring the cGFP signal. Both inoculation assays showed that F. graminearum abundance was reduced when preinoculated with F. poae, while F. poae benefitted from the presence of F. graminearum, resulting in a small but consistent increase in its biomass compared to a single F. poae inoculation. We assumed that F. poae benefits from the increased amount of damaged tissue caused by the aggressive pathogen F. graminearum to expand on wheat. To confirm our hypothesis, we inoculated the GFP‐tagged F. poae on mechanically wounded leaves. F. poae showed more extensive growth in larger wounds, advocating a more saprophytic role for F. poae in the disease complex. It should be noted that in this study we used single isolates of F. graminearum and F. poae, which means that the obtained results may be isolate‐specific.

In a paper by Pirgozliev et al. (2012) the interaction between Microdochium spp. and F. culmorum was investigated, and they reported on increased FHB symptoms and DON accumulation when wheat heads were preinoculated by the more saprophytic Microdochium spp. followed by F. culmorum. Because DON is produced on stress, it was suggested that the competition for space and nutrients attributed to the increase in DON resulted in increased FHB. On the other hand, Xu et al. (2007) found no synergistic interactions for FHB pathogen coinoculations. However, competition between Fusarium isolates was reported in which F. graminearum was most competitive in dual inoculation with F. poae, F. culmorum, and F. avenaceum, where these latter pathogens had significantly less fungal biomass compared to single inoculations. More recently, Siou et al. (2015) highlighted a possible interaction between F. poae and F. graminearum, stating that the colonization of wheat by F. poae in the centre of the ear hampered the infection by F. graminearum inoculated three spikelets higher. This study highlighted the possible antagonistic effects of F. poae against F. graminearum, but did not further investigate the underlying mechanisms.

To explain the observations from this study, we first assessed the direct antagonism between F. poae and F. graminearum by coculturing them in vitro. However, coinoculation of both species on PDA plates did not uncover any mutual antagonism (Figure S5). Although different conditions were used, this observation was in slight contrast to previous reports showing that the weak pathogen F. poae did not influence the in vitro growth and germination of F. graminearum while the latter one outcompeted F. poae in a confrontation assay (Wagacha et al., 2012).

We further explored the interaction between F. poae and F. graminearum, hypothesizing that F. poae and F. graminearum interact during the infection of wheat at the level of their trichothecenes. This hypothesis originates from the fact that DON is a crucial virulence factor during the spread of F. graminearum in the rachilla and the rachis (Audenaert et al., 2014). In addition, it is known that F. poae produces a much more diverse blend of mycotoxins comprising both type A and type B trichothecenes (Vanheule et al., 2017). Therefore, we wanted to investigate whether precolonization of wheat ears or detached leaves with the weak pathogen F. poae resulted in accumulation of F. poae‐specific mycotoxins like NIV, DAS, and NEO, which in turn could affect the growth and colonization of F. graminearum. However, none of the F. poae trichothecenes could be detected during the colonization of detached leaves or wheat ears, despite the fact that in previous research we have already shown isolate 2,516 to be a DAS and NEO producer (Vanheule et al., 2017), which suggests that these toxins serve other purposes and are not involved in the competition for niche in the FHB disease complex. The situation was different for F. graminearum, as preinoculation of F. poae resulted in a reduced presence of 15‐ADON and DON compared to single F. graminearum inoculations. This reduced amount of type B trichothecenes in mixed inoculations could be attributed to a lower amount of F. graminearum biomass but not to a reduced production per biomass.

As trichothecene production could not explain the antagonistic effect of F. poae preinoculation on colonization by F. graminearum, we hypothesized that a plant‐mediated response induced by preinoculation of F. poae might hamper a later F. graminearum infection. The activation of plant defence in tomato by Fo47, a nonpathogenic strain of F. oxysporum against a pathogenic F. oxysporum strain, has already been demonstrated (Fuchs et al., 1997; Aimé et al., 2013). Moreover, this was not only limited to strains from the same species of a single genus as Fo47 could also protect against Pythium ultimum in cucumber by inducing host plant defence responses (Benhamou et al., 2002).

Pursuing a more holistic approach, we combined multispectral and gene expression data in a PCA. By monitoring crucial wheat defence genes, we showed that a single F. graminearum infection and coinoculation of F. graminearum and F. poae resulted in a consistent down‐regulation of SA or JA defence pathways through down‐regulation of LOX and ICS genes. In contrast, and although F. poae does not induce symptoms, a sequential up‐regulation of ICS and LOX genes was observed at several time points after the inoculation. We hypothesize that the early induction of SA‐ and JA‐related defences by F. poae hampers a subsequent F. graminearum infection. Previously, we have shown, using a priming approach with the green leaf volatile Z‐3‐hexenyl acetate, that a successful defence of wheat against F. graminearum is sequentially and meticulously regulated by SA and JA during the early stages of infection (Ameye et al., 2015). Similarly, preinoculation of detached leaves or wheat ears with a weak pathogen such as F. poae modifies this meticulously regulated system of the plant's innate defence. Similar results were obtained in a different study in which we investigated an interkingdom interaction between wheat, F. graminearum, and Sitobion avenae (English grain aphid; De Zutter et al., 2017). In that work, preinfestation of wheat ears with aphids resulted in down‐regulation of PAL at early time points, which resulted in suppression of PAL during a subsequent F. graminearum infection compared to a single F. graminearum infection and a more proliferated outgrowth of the fungal pathogen (De Zutter et al., 2017).

Together our results highlight the importance of temporal dynamics between different members of the same disease complex in that the timing of infection shapes the outcome of symptom development and pathogen survival by extension.

4. EXPERIMENTAL PROCEDURES

4.1. Fusarium spp. survey

The FHB population on winter wheat was surveyed during all growing seasons between 2002 and 2018 in seven locations situated in the main wheat‐growing regions of Flanders. In each growing season a total of 10 popular commercial varieties were included in the FHB survey, using a randomized block design with at least three replications providing a total of 210 fields per year, where plot size varied between 15 and 25 m². The locations of these fields are reported in Audenaert et al. (2009). Conventional crop management was practised, including three nitrogen fertilizations according to the N‐index established by the soil service of Belgium (Geypens et al., 1994) and pre‐ and postemergence herbicide applications. One fungicide treatment was applied at growth stage (GS) 59. The fungicide treatment (epoxyconazole [75 g/ha] and kresoxim‐methyl [125 g/ha]) was not targeted against Fusarium spp. but was included to control other leaf and ear diseases.

Two weeks after anthesis, two diseased ears were selected per field for assessing the presence of Fusarium spp., which resulted in 420 samples per year. Individual spikelets with symptoms were plated and outgrowing fungi and monospore isolates were prepared. The species used in present study were identified by PCR and/or EF1 sequencing as previously described in Audenaert et al. (2009).

4.2. Fusarium strains and transformation of F. graminearum and F. poae

F. graminearum PH‐1 (Trail and Common, 2000) and F. poae 2,516 (Vanheule et al., 2016) were used throughout the study. The GFP‐encoding gene together with promoter was amplified from plasmid pRFHUE‐GFP (addgene: #89469). The amplified fragments were inserted after hygromycin B of the plasmid vector pII99 between the cut sites of restriction enzymes HindIII and SacI (Thermo Scientific; Figure S16). Transformation of F. graminearum and F. poae was adjusted from Ammar et al. (2013) and performed as described in Methods S1. After transformation, the fitness of the transformant was assessed at the level of vegetative growth and sporulation, and additional virulence assessed for F. graminearum. No differences were observed when comparing with the wild type (Figure S17).

4.3. Fungal growth, production, and isolation of conidia

The GFP transformants of F. graminearum PH‐1 or F. poae 2,516 were grown on PDA for 7 days at 21°C under a regime of 12 hr of dark and 12 hr of combined UVA and UVC light (2 × TUV 8 W T5 and 1 × TL 8 W BLB; Philips). The transformants of F. graminearum PH‐1 and F. poae 2,516 had no difference in fitness and/or virulence compared to the wild‐type strains (Figure S17). Conidia were harvested by adding a solution of sterile 0.01% (vol/vol) Tween 80 to the PDA plates and rubbing the mycelium with a Drigalski spatula. Four layers of sterile Miracloth were used to filter the conidia and remove the mycelium. The suspension was diluted to a final concentration of 10⁶ conidia/ml. This suspension was used for inoculations on both wheat leaves and ears.

4.4. Plant materials

For the leaf bioassay, spring wheat cv. Tybalt was grown in pots (4.5 cm diameter × 6.5 cm height) in a growth chamber (21°C, 16 hr:8 hr, light:dark) for 10 days. For the wheat ear bioassay, six spring wheat cv. Tybalt were grown in each pot (15 cm diameter × 30 cm height) under glasshouse conditions until GS 65.

4.5. Detached leaf and ear inoculation assays

Leaf segments of 4 cm were cut from the tip of the leaves of 10‐day‐old seedlings. These leaves were placed on their abaxial surface in Petri dishes containing 0.5% (wt/vol) bacteriological water agar amended with 40 mg/L benzimidazole. The conidial suspension (2.5 µl) was deposited in the centre of the leaf segment, which was wounded using a sterile inoculation needle as previously described (Ameye et al., 2015).

Two parallel experiments were performed (Figure S18). (a) To assess the impact of F. poae on the infection by F. graminearum, detached leaves were infected with wild‐type F. poae 1 day before F. graminearum infection (p[−1d] + g) or simultaneously infected with F. poae and F. graminearum (g + p). In control treatments F. poae was replaced with water, w(−1d) + g and w + g. (b) To assess the impact of F. graminearum inoculation on the F. poae infection, detached leaves were infected with F. graminearum 1 day before F. poae infection (g[−1d] + p) or simultaneously infected with F. poae and F. graminearum (g + p). In the control treatments, instead of an inoculation with F. graminearum, water was used: w(−1d) + p and w + p, respectively. The disease progress in the detached leaves was assessed using a multispectral phenotyping platform including excitation LEDs to visualize and quantify GFP for 3 days. To assess the impact of F. graminearum on the growth of F. poae, detached leaves were inoculated with GFP‐tagged F. poae and wild‐type F. graminearum. GFP‐tagged F. poae was also inoculated on leaves that were mechanically wounded by scratching the epidermal layer over a length of 0.1, 1, and 2 cm with a sterile scalpel.

The same approach was pursued in the wheat ear trials using the same nomenclature. At GS 65, ears were inoculated with F. graminearum, F. poae, or a combination of both at different time points. We used a point inoculation strategy in which one spikelet of each ear was infected with 10 µl of conidial suspension. The disease progress was monitored using a multispectral phenotyping platform for 7 days. Visual scoring assessment of ears inoculation was performed at 7 days after inoculation (dai) using a five‐class disease index, where level 1 = no symptoms visible, level 2 = one ear caryopsis has symptoms, level 3 = two ear caryopses have symptoms, level 4 = all ear caryopses on the inoculation site have symptoms, and level 5 = all ear caryopses have symptoms (Figure S19).

4.6. Disease progress: multispectral phenomics

To assess disease progression in detached leaves and in wheat ears, we employed a custom‐build multispectral phenotyping and microdispenser platform called the PathoViewer. This platform allows the visualization of diverse physiological traits in real time, based on specific absorption, reflection, and emission patterns at a high temporal and spatial resolution at 6 µm. A monochrome camera system, including a filter wheel, allows pixel‐to‐pixel capturing of RGB values, chlorophyll fluorescence (Chl), and GFP fluorescence. (CropReporter; Phenovation). This camera system was mounted on a Cartesian coordinate robot that was housed in an acclimatized environment of temperature, light, and humidity. The chamber was equipped with sun LED modules (SLMs) (Phenovation).

Fungal progress was assessed based on symptom development using the RGB image. In addition, the effect of disease on the efficiency of photosystem II (F_V/F_M) was assessed (Baker, 2008). Finally, the GFP signal of F. graminearum or F. poae was used as a hallmark for fungal presence. GFP signals were corrected for autofluorescence of senescing leaves and ears resulting in the corrected GFP value (cGFP).

Using this approach, images were taken on 1, 2, and 3 dai in the detached leaf assay. As the disease progress and symptom development were slower in the ears, images were taken on 1, 2, 4, and 7 dai.

Secondary metabolite production was assessed based on the production of the red pigment rubrofusarin, which is typically synthesized by the gene cluster consisting of the gene coding for polyketide synthase PKS12, aurJ, aurF, gip1, and the hypothetical gene FG02329.1 (Frandsen et al., 2006).

4.7. RNA extraction and RT‐qPCR

Total RNA from both detached leaves and ears was extracted using TRIzol reagent (Sigma‐Aldrich) according to the manufacturer's instructions and subsequently quantified with a Quantus fluorometer (Promega). First‐strand cDNA was synthesized using the GoScript reverse transcription system (Promega). Using GoTaq qPCR Master Mix (Promega), RT‐qPCR analysis was performed using a CFX96 system (Bio‐Rad) with the following thermal settings: 95°C for 2 min, 40 cycles of 95°C for 15 s, and 60°C for 1 min. Melting curve analysis was performed using a temperature profile of heating to 95°C at the rate of 0.5°C/s. The primers used for all genes are listed in Table S2. Normalization of defence genes was performed using the cell division control protein gene (Ta54227) in wheat as reference. Fungal biomass was quantified using a pre‐mRNA slicing factor of F. graminearum (FGSG_01244) and F. poae (FPOA_01282) as described before (Ameye et al., 2015). TRI5 gene detection primers designed specifically for F. graminearum (FGSG_TRI5) and F. poae (FPOA_TRI5) confirmed no cross‐reactivity in the PCR. Gene expression analysis was performed using qBase + software (Biogazelle).

4.8. Quantification of type A and type B trichothecenes in detached leaves and wheat ears

To investigate whether coinoculation of F. poae and F. graminearum on wheat seedlings and ears impact the mycotoxin production, 10 different mycotoxins produced by both F. poae and F. graminearum were screened using LC‐MS/MS based on De Boevre et al. (2012) and the methods described in Methods S2.

4.9. Statistical analysis

For statistical evaluation, PCA and plots generation, the R software v. 3.6.0 (R Core Team, 2019) and the packages ggplot2 (Wickham, 2016), factoextra (http://www.sthda.com/english/rpkgs/ factoextra), agricolae (de Mendiburu, 2014), carData, car (Fox and Weisberg, 2018), FSA (Ogle, 2017), and cooccur (Griffith et al., 2016) were used. For multiple comparisons, the normality and homogeneity of variances was assessed by the Shapiro–Wilk and Levene tests, respectively. When conditions were met, an analysis of variance (ANOVA) was performed followed by a post hoc Tukey test. When the conditions for parametric analyses were not met, a nonparametric Kruskal–Wallis test was used followed by a pairwise comparison using a corrected Dunn's test. All analyses were run at a significance level of α = 0.05. Two‐way interactions and higher order interactions between Fusarium species were analysed using a simple straightforward log‐linear model as described by Xu et al. (2005) assuming a Poisson sampling in each location. K‐way and higher‐order effects were determined to significance levels of α = 0.05.

Supporting information

FIGURE S1 Impact of Fusarium poae 2516 inoculation on F. graminearum PH‐1 infection in a detached leaf assay at 4 days after inoculation

FIGURE S2 Impact of Fusarium poae 2516 inoculation on F. graminearum PH‐1 infection in a detached leaf assay at 1 to 3 days after inoculation

FIGURE S3 Impact of Fusarium poae 2516 on pigmentation by F. graminearum PH‐1

FIGURE S4 Mock inoculation with water on wheat leaves

FIGURE S5 Interaction between Fusarium graminearum PH‐1 and F. poae 2516 on potato dextrose agar plate

FIGURE S6 Impact of Fusarium graminearum PH‐1 inoculation on F. poae 2516 infection in a detached leaf assay at 4 days after inoculation

FIGURE S7 GFP‐tagged Fusarium poae 2516 inoculation on leaf wounds from 1 to 4 days after inoculation (dai)

FIGURE S8 Impact of Fusarium poae 2516 inoculation on F. graminearum PH‐1 infection in a detached leaf assay

FIGURE S9 Impact of Fusarium poae 2516 inoculation on F. graminearum PH‐1 infection on wheat ears

FIGURE S10 Impact of Fusarium poae 2516 on mycotoxins produced by F. graminearum PH‐1

FIGURE S11 Expression profile of marker genes for salicylic acid and jasmonate biosynthesis, peroxidase, and pathogenesis‐related proteins in wheat leaves infected with Fusarium poae 2516 and F. graminearum PH‐1

FIGURE S12 Expression profile of marker genes at 1, 2, 4, and 7 days after inoculation on ears

FIGURE S13 Expression profile of marker genes in the inoculation of Fusarium poae after F. graminearum on detached leaves

FIGURE S14 Principal component analysis of gene expression, fungal biomass corrected GFP value, normalized quantitative relative values, and the health condition of the plant (F_V/F_M, efficiency of photosystem II) from 1 to 3 days after inoculation and all time points in the leaf assay

FIGURE S15 Principal component analysis of gene expression, fungal biomass corrected GFP value, normalized quantitative relative values, and the health condition of the plant (F_V/F_M, efficiency of photosystem II) at 1, 2, 4, and 7 days after inoculation in ear assay

FIGURE S16 Construction of the vector expressing green fluorescent protein (GFP)

FIGURE S17 Fitness of the green fluorescent protein (GFP) transformant and wild type

FIGURE S18 Setup of the inoculation

FIGURE S19 Visual scoring assessment of ears inoculation

TABLE S1 Data of the total explained variation of principal component analysis

TABLE S2 Primers used for quantitative reverse transcription PCR

METHODS S1 Transformation of Fusarium poae 2516 and F. graminearum PH‐1

METHODS S2 Quantification of type A and type B trichothecenes in detached leaves and wheat ears

Tan J, Ameye M, Landschoot S, et al. At the scene of the crime: New insights into the role of weakly pathogenic members of the fusarium head blight disease complex. Molecular Plant Pathology. 2020;21:1559–1572. 10.1111/mpp.12996

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.