Introduction
Trichoderma ear rot disease in maize was first described in 1973 (Shurtleff et al., 1973). The Compendium of Corn Diseases considers several fungi as “secondary invaders”, including Trichoderma spp., which often cause disease after severe leaf damage induced by other fungi or ear damage from Helminthosporium maydis infection. Disease caused by Trichoderma viride is listed as “other corn ear rots” and is associated with injury to the developing ear (White, 1999). Several publications associate Trichoderma spp. infections with other leaf or ear diseases, being related to damage (by weather conditions or insects) to the developing ear that provides access to windblown spores and rainfall (Vincelli, 2014; Wise et al., 2016; OSU, 2020). In short, diseases caused by Trichoderma spp. are sporadic, scattered within a field, and only occur when there is previous damage to the plant (from insect feeding and heavy storms). However, it has recently been reported that very aggressive strains of Trichoderma afroharzianum are the primary agent producing ear rot disease in the warmer regions of southern Germany and France (Pfordt et al., 2020b) as well as Italy (Sanna et al., 2022).
According to Wise et al. (2016), ear rot fungi associated with mycotoxins include Aspergillus flavus, Fusarium verticillioides (primary fungus causing Fusarium ear rot), as well as Gibberella zeae (syn. Fusarium graminearum), Diplodia maydis (syn. Stenocarpella maydis), and Diplodia macrospora (syn. Stenocarpella macrospora) in specific regions but not in others, suggesting a significant reliance on environmental factors. Therefore, Penicillium is capable of producing mycotoxins only under conditions of high humidity, and regarding Trichoderma, it appears that only certain species have been observed to produce mycotoxins on previously damaged maize kernels.
Before the widespread use of molecular techniques, it was difficult to identify the isolates of Trichoderma spp. based solely on morphological traits. Thus, the scientific literature is full of misleading identifications of Trichoderma. Druzhinina et al. (2005) set up the basis for correct Trichoderma identification through the use of advanced molecular tools. Trichoderma spp. are among the most common filamentous fungi isolated from soil, rotting wood, other fungi, and innumerable substrates (Druzhinina et al., 2011; Kubicek et al., 2011). Moreover, Trichoderma rhizosphere-competent strains interact with plants, increasing mineral uptake and activating the plant immune system with the subsequent increase of plant growth and resistance to a range of pathogens and abiotic stress (Yedidia et al., 1999; Gupta and Bar, 2020). Both induced systemic resistance (ISR) and systemic acquired resistance (SAR) have been described in Trichoderma–plant root interactions; the signaling molecules include salicylic acid (SA), jasmonic acid, and ethylene (Pieterse et al., 2014; Kubicek et al., 2019). The physical interaction of Trichoderma–plants is limited to the root epidermis, primary cell layers, and outer cortex, requiring a temporary suppression of SA-dependent plant defenses (Yedidia et al., 1999; Alonso-Ramírez et al., 2014; Poveda et al., 2020). A recent study on the coevolution of the interactions of plant–Trichoderma spp. using liverwort, pteridophyte, and angiosperm models suggested that the fungus behaved as a pathogen until plants developed the defense system based on SA, thus limiting its colonization (Poveda et al., 2023).
Samuels and Hebbar (2015) recorded Trichoderma populations ranging from 102 to 103 spores/g of soil or root, while Wolna-Maruwka et al. (2017a) found natural populations of Trichoderma harzianum and Trichoderma atroviride occurring at 3.4 × 102 spores/g of dry soil in field experiments in Poland. In another study, Wolna-Maruwka et al. (2017b), again only counting T. harzianum and T. atroviride, found 0.36 × 102 spores/g of dry soil in the control samples from their field experiment. Oskiera et al. (2017), using a newly developed multiplex PCR technique, identified 103–104 CFU/g of naturally occurring Trichoderma spp. in Polish soil collected from a field experiment.
The biodiversity of the genus Trichoderma (teleomorph Hypocrea, Ascomycota) has been studied in several publications. Interestingly, Jaklitsch and Voglmayr (2015) classified 650 species of Trichoderma from different European climate regions using tef1 sequence data. The tef1 gene is considered the most useful reference gene to identify and distinguish the different Trichoderma species (Samuels and Hebbar, 2015). In the publication discussion of Pfordt et al. (2020b), some concerns were raised about the potential production of mycotoxins by aggressive strains of T. afroharzianum. However, Degenkolb et al. (2008) considered the Brevicompactum clade a separate lineage in Trichoderma/Hypocrea that contains Trichoderma brevicompactum and the new species Trichoderma arundinaceum, Trichoderma turrialbense, Trichoderma protrudens, and Hypocrea rodmanii. With the exception of H. rodmanii, all members of this clade produce trichothecene-type toxins. Degenkolb et al. (2008) also re-identified the trichothecene-producing T. harzianum, T. viride, and Hypocrea sp. as T. arundinaceum. In several publications, including Kubicek et al. (2019), Trichoderma reesei has been recently recognized as the model organism for the industrial production of cellulolytic enzymes. Other Trichoderma spp. are opportunistic species in mushroom farms (Trichoderma aggressivum) or can become pathogens in immunocompromised people (Trichoderma longibrachiatum) (Komon-Zelazowska et al., 2007; Druzhinina et al., 2008). Degenkolb et al. (2008) stated that the species in the Brevicompactum clade are not closely related to the Trichoderma species that have biological applications in agriculture.
In Europe, for a microorganism strain (active substance) to be registered as a microbial plant protection product (PPP), it should demonstrate that it is safe for humans and the environment. According to the EU Regulation 1107/2009, several studies should be performed to prove that the PPP does not produce/contain any toxic metabolite of human concern. Risk assessment is also required to indicate the background levels of the product in the environment (air, water, and soil) after field/greenhouse application at the recommended doses and use pattern in the crop and after harvest. To be able to authorize a product as PPP for use in certain crops/diseases, Good Experimental Practice (GEP) studies are required [according to the standards of the European and Mediterranean Plant Protection Organization (EPPO)] to prove the product’s efficacy and the absence of phytotoxicity to the plants on which it is being applied. Furthermore, other observed benefits may be claimed, such as yield increases and reductions in mycotoxin contents, for example, if such effects are observed during the GEP efficacy trials. Three doses as well as the mode of application (on seeds, soil/substrate, drip/air irrigation, etc.) of the authorized PPP are required by the authorities to grant authorization to a product. The dossier on the proposed PPP is assessed and reviewed by member states and the European Food Safety Authority (EFSA) before being finally approved or not by the EU Commission (Trillas et al., 2020).
The disease triangle
Environmental and agronomic practices and field site
As described in all plant pathology books, the disease triangle is important to assess disease. In addition to the pathogen and the plant host, the environment (temperature, relative humidity, and precipitation) are key factors in the plant disease triangle. Such information was not reported in detail in the work from Pfordt et al. (2020b) to assess the environmental conditions and the management practices of the crop from where the Trichoderma isolates were obtained. The publication briefly cited the growing conditions of the maize: “symptoms observed in Southern Bavaria, after warm and dry summer”. However, this information and the agronomic practices were already known because research on complex Fusarium ear rot was conducted from 2016 to 2018 in 58 locations in Germany, including the same fields from where the isolates of the Trichoderma spp. were obtained (Pfordt et al., 2020a). In these fields, Pfordt et al. (2020b) recorded the mean temperature and precipitation in July (during flowering when the impact of Fusarium disease was the highest) but did not report on the climatic condition during the growing season and the insect pest management strategy used. Furthermore, information on the temperature (23°C) under seasonal day/night light cycles was given for the greenhouse experiments assessing the pathogenicity of the Trichoderma strains, but all the other environmental conditions were not mentioned.
Co-occurrence of <italic>Trichoderma</italic> and <italic>Fusarium</italic>
Pfordt et al. (2020b) mentioned that “surprisingly” in 2018, a “severe occurrence” of Trichoderma on maize was recorded at “a field site” in Southern Germany, but its specific location was not provided. This field has been also used to study a Fusarium species complex, using artificial inoculation of the pathogen, which is detailed in the supplementary materials of Pfordt et al. (2020a, 2020c). The level of disease indicated by “severe occurrence” could have been specified more accurately as the number of cobs with Trichoderma ear rot compared to the uninfected controls or in relation to other Fusarium ear rot diseases or maize plants with Fusarium disease. Fusarium diseases are not mentioned at all in Pfordt et al. (2020b), where the Trichoderma spp. may be associated with Fusarium spp. mycoparasitism. There is evidence that endophytic Fusarium species cause symptomless infections in kernels (Gromadzka et al., 2019), with the incidence of symptomless Fusarium infections being higher than that of kernel rot. The finding that Fusarium can be endophytic is well documented in several publications, which report that the fungus is found in the embryo and endosperm of kernels and is associated with animal toxicity (Bacon et al., 1992; Yates et al., 1997). The presence of Fusarium subglutinans and F. verticillioides together with T. atroviride was previously reported in maize ears with significant Fusarium ear rot in Poland from 2014 to 2017 (Gromadzka et al., 2019). In fact, studies performed in Poland (Gromadzka et al., 2016) indicate the co-occurrence of Fusarium spp. with other fungi in the same kernel, with T. atroviride being the most abundant species (31%). Consequently, our opinion is that in opposition to what is claimed by Pfordt et al. (2020b), Trichoderma spp. are present in the maize ear rot not as a primary pathogen but in co-occurrence with Fusarium spp. as described in several studies in Poland (Gromadzka et al., 2019).
Maize plants
Pfordt et al. (2020b) should have mentioned the maize hybrids used in the greenhouse experiments (The Methodology) to test the pathogenicity of the Trichoderma isolates. It has only been stated that “maize seeds of two varieties” were used. Moreover, in the Introduction, there is no mention of the maize hybrids from where the Trichoderma spp. isolates were obtained, other than stating that the cobs of “20 maize varieties” were sampled. In Pfordt et al. (2020c), it is mentioned that four susceptible maize varieties were used for the inoculation with different Fusarium species to evaluate their pathogenicity and mycotoxin production. It is known that the development of maize hybrids resistant to Fusarium ear rot is important to minimize the risks of mycotoxin (Pascale et al., 2002). For example, the use of maize hybrids that are genetically engineered with the genes expressing the cry toxin of Bacillus thuringiensis is important since the incidence of symptomless Fusarium infections in kernels is reduced when compared with the near-isogenic hybrids (Munkvold et al., 1997).
The methodology
Greenhouse study to assess pathogenicity
In Pfordt et al. (2020b), Table 1, isolate Tri1 from France as well as isolates Tri2, Tri3, Tri4 (2018), and Tri5 (2019) from Germany were obtained from the plants with symptoms of Trichoderma ear rot), while the other 15 isolates tested were obtained from different sources. In Pfordt et al. (2020b), Table 2, after artifical inoculation, significant differences in disease severity (lowest disease levels) were observed for T. harzianum T39 (commercial microbial PPP) and T. harzianum T12 (university strain with potential biocontrol activity). The highest disease severity was observed for the T. afroharzianum Tri1, Tri2, Tri3, and Tri5 isolates. No disease was observed in the greenhouse study with Tri4 (Trichoderma tomentosum), which had been isolated from cobs showing symptoms of Trichoderma disease, or with the reference type strain T. afroharzianum CBS124620, which had been isolated in Peru from Theobroma cacao plants. The fact that no disease was produced by the reference type strain was attributed to the possible loss of pathogenicity, while no comments were made about the non-pathogenicity of T. tomentosum. Since silk channels have a complex and dynamic microbiome that is rich in nutrients (Khalaf et al., 2021), another possibility is that the reference type strain may prefer different carbon sources. T. tomentosum and T. harzianum remain in the Green/Harzianum clades (Jaklitsch and Voglmayr, 2015), while T. harzianum and T. afroharzianum belong to the Harzianum/Virens clades (Kubicek et al., 2019). They do not belong to the Brevicompactum clade.
The strains associated with <italic>Trichoderma</italic> ear rot in Europe
T. atroviride was reported to occur in 14% of the maize samples examined between 2014 and 2017 in Poland (Gromadzka et al., 2019). Earlier studies by the same research group (Blaszczyk et al., 2011; Blaszczyk et al., 2017) reported that T. atroviride accounted for a minor proportion of the isolates obtained from samples, with T. harzianum being the prevalent species in Poland. It is worth noting that Gromadzka et al. (2019) also reported that competitive species of T. atroviride reduced the mycotoxin content in maize samples. Other Trichoderma spp. also reduce mycotoxins in vitro and in planta (Modrzewska et al., 2022; Dini et al., 2022). Sanna et al. (2022) classed two strains of T. afroharzianum as being responsible for seed rot in maize in Italy by also using artificial inoculation experiments based on the method described by Pfordt et al. (2020b).
Artificial inoculation
The silk channel inoculation method is widely used to evaluate resistance/susceptibility to Fusarium ear rot (Mestarhazy et al., 2020). Other methods have not been compared and may be more appropriate, such as the needle pin and toothpick or spray-pulverization technique. Pfordt et al. (2020c) tested two spore densities and two inoculation methods in the pathogenicity tests on maize cobs in field conditions, using a spore density of 104 spores/mL for F. graminearum and 106 spores/mL for the other Fusarium spp. Their findings indicated that the aggressiveness of F. graminearum may be higher than that of the other Fusarium species. In the assay used by Pfordt et al. (2020b), to assess pathogenicity, there was no justification for using such a high concentration of spores of 106 conidia/mL of Trichoderma. Such high concentrations of Trichoderma are extremely unlikely to occur in the air and have never been described in the literature. Moreover, saprotrophy is a very ancient trait that is widespread in the Trichoderma genus and fungi and could also occur in the rhizosphere and soil. Mycoparasitism is also found, but only a few species of Trichoderma have been isolated as endophytes (Druzhinina et al., 2011). The most plausible scenario is that local Trichoderma spp. associated with the saprotrophy and mycoparasitism of Fusarium spp. were responsible for the observations reported by the authors.
Author contributions
MT: Conceptualization, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing, Data curation, Investigation, Methodology, Validation. GS: Conceptualization, Validation, Writing – original draft, Writing – review & editing, Data curation, Investigation, Methodology, Supervision. MA: Writing – review & editing, Data curation, Investigation, Methodology, Validation, Conceptualization, Supervision, Writing – original draft.