Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2020 Aug 31;5(36):23276–23283. doi: 10.1021/acsomega.0c03090

In Vitro Antagonistic Activity of Trichoderma harzianum against Fusarium sudanense Causing Seedling Blight and Seed Rot on Wheat

Silvina Larran †,*, M Pilar Santamarina Siurana ‡, Josefa Roselló Caselles ‡, María Rosa Simón §,⊥, Analía Perelló †,∥
PMCID: PMC7495787  PMID: 32954178

Abstract

graphic file with name ao0c03090_0007.jpg

Fusarium sudanense is a novel fungus recently isolated from asymptomatic samples of wheat grains in Argentina. The fungus caused symptoms of seedling blight and seed rot on wheat after artificial inoculations. It is known that the production of mycotoxins by pathogens belonging to the Fusarium genus is harmful to human and animal health. Moreover, the warm and humid conditions that are favorable for growth and mycotoxin production of these species put the Argentinian wheat production area at a high risk of mycotoxin contamination with this novel pathogen. The aim of this work was to evaluate the antagonistic effect of Trichoderma harzianum against F. sudanense under in vitro tests at different environmental conditions. Fungi were screened in dual culture at different water activities (αw) (0.995, 0.98, 0.95, and 0.90) and temperatures (25 and 15 °C). The growth rate of the fungi, interaction types, and dominance index were evaluated. Also, the interaction between T. harzianum and F. sudanense was examined by light and cryo-scanning microscopy. T. harzianum suppressed the growth of F. sudanense at 0.995, 0.98, and 0.95 αw at 25 °C and 0.995 and 0.98 αw at 15 °C. Macroscopic study revealed different interaction types between F. sudanense and T. harzianum on dual culture. Dominance on contact where the colonies of T. harzianum overgrew the pathogen was the most common interaction type determined. The competitive capacity of T. harzianum was diminished by decreasing the temperature and αw. At 0.95 αw and 15 °C, both fungi grew slowly, and interaction type “A” was assigned. Microscopic analysis from the interaction zone of dual cultures revealed an attachment of T. harzianum to the F. sudanense hyphae, penetration with or without formation of appressorium-like structures, coiling, plasmolysis, and a veil formation. According to our results, T. harzianum demonstrated capability to antagonize F. sudanense and could be a promising biocontrol agent.

Introduction

Current agriculture faces several global problems linked with its intensification and loss of biodiversity among other factors, which led to a forced search to look for more sustainable long-term alternatives. Accordingly, it is imperative to investigate more efficient management of available resources, which may result in benefits from environmental and economic points of view. One of these alternatives is to use fungi and their metabolites in the biological control of plant diseases; some of which produce secondary metabolites used to defend plants that are a very important source of chemical diversity.1,2 There is still much to explore about metabolism, ecology, and the role of bioactive fungi in the expression of diseases in important crops like cereals. This implies a very broad and interesting field to search for secondary metabolites with various activities applicable as a potential source of bio-input as part of the Integrated Disease Management Strategies in Plant Health.3,4 This potential provides us with a new tool in the current search for sustainable alternatives for agricultural pest management using new environmentally friendly bioproducts. That is, unlike synthetic chemical fungicides, their use does not represent negative effects, such as water and soil contamination or toxicity on pollinating insects, animals, or humans.

Fusarium sudanense S.A. Ahmed, Al-Hatmi & de Hoog is a novel species in the “Fusarium fujikuroi species complex” (FFSC)5 that was isolated from asymptomatic samples of wheat grains in Argentina causing seedling blight and seed rot on wheat.6 Thus, according to recent references, this suggests that the fungus could be a pathogen in a latent phase.7 It is also known that latent pathogens may produce disease symptoms in certain plant growth stages or under certain environmental or nutritional conditions.

According to Shi et al.,8Fusarium spp. infects cereal crops in different regions, which causes major yield losses or reduces crop quality worldwide. In wheat diseases, fungi are one of the major limiting factors for the wheat yield and quality.9 In particular, an asymptomatic period has been registered in the hosts of some species in the Fusarium genus. However, these fungi can break latency and switch to a pathogenic lifestyle, which induces symptoms in their hosts.10 Additionally, the production of mycotoxins by pathogens belonging to the Fusarium genus with severe risks to human and animal health, has been reported by different authors.11,12 Interestingly, mycotoxins produced by fungi belonging to the genus Fusarium continue to be discovered on widely known species and/or on novel species.13

F. sudanense on wheat grains in Argentina is a new phytosanitary risk that can cause significant losses during the implantation of the crop. The Argentine Pampas is one of the most highly productive areas of food in the world and currently contributes 2% of wheat global production. Moreover, the country is among the main exporters of grain and products from this crop in the world market.14 However, despite an increased understanding of mycotoxigenic Fusarium spp. and mycotoxins in Argentina, knowledge on practices and measures to control both the fungus and its toxins is very limited.15 For this reason, we focused on new innovative eco-friendly strategies for its control to minimize yield losses and prevent or reduce possible mycotoxin production. Accordingly, biological control by employing beneficial fungi helps reduce environmental contamination and resistance caused by the application of synthetic chemical products. In recent years, many research studies conducted in Argentina have focused on developing alternatives to chemicals for sustainable wheat disease management, such as using biopesticides.15 In agreement with this new paradigm for disease control, this work aims to evaluate the biological control of F. sudanense with a fungal strain belonging to the Trichoderma genus, which was extensively studied as an antagonist against different important phytopathogens.16−21 Interestingly, previous studies conducted in Valencia (Spain) have recorded that Trichoderma harzianum (CECT 20736) successfully antagonizes several rice pathogens by in vitro assays.22

It is also known that both biotic and abiotic factors, such as temperature and moisture, affect fungal growth.23 Previous ecophysiological studies in F. sudanense have demonstrated that fungus growth is affected by temperature and water availability in culture media.4 However, the impact of such factors for F. sudanense in the presence of potential antagonists is currently unknown. Therefore, the impact of the main environmental requirements for fungal growth will provide a better understanding of its behavior in nature when it interacts with other fungi.

The objectives of the present work were to (i) evaluate the effectiveness of T. harzianum (CECT 20736) against F. sudanense under different water activities and temperature conditions, (ii) macro- and microscopically determine the interaction types between both species under different conditions, and (iii) analyze the mechanisms of action involved in antagonizing the pathogen using light microscopy and cryo-scanning electron microscopy.

Results

Fungal Interaction in the Dual Culture

F. sudanense and T. harzianum growth was affected by water activity and temperature as shown in Table 1 for the single factors. Considering the main factors, T. harzianum had a higher growth rate compared to F. sudanense. The average of the growth rates of both species increased at 25 °C compared to 15 °C (an average of 6.50 and 1.94 mm/day respectively, not shown) and increased with the increment in the water activity.

Table 1. Analysis of Variance on the Growth Rates of F. sudanense and T. harzianum at Four Water Activities (αw), Two Temperatures, and Their Interactions.

source of variation DFa MSb P valuec
repetition 3 0.015  
species 1 135.344 ≤0.001
temperature 1 331.467 ≤0.001
αwd 3 269.349 ≤0.001
species and temperature 1 10.232 ≤0.001
species and αw 3 67.040 ≤0.001
temperature and αw 3 60.400 ≤0.001
species and temperature and αw 3 10.835 ≤0.001
species and temperature and αw 3 0.072 0.358
residual 31 0.065  
Open in a new tab
a

DF: degrees of freedom.

b

MS: mean squares.

c

Fisher test (P ≤ 0.05).

d

αw: water activity.

However, double interactions species × temperature; species × water activity; temperature × water activity and the triple interaction species × temperature × water activity were also significant (Table 1). The significant species × temperature × water activity interaction is represented in Figure 1. At 15 °C, T. harzianum had significantly higher growth rate at 0.98 and 0.995 αw than F. sudanense, whereas at 0.95 and 0.90 αw, no statistically significant differences were detected according to the LSD (P ≤ 0.05) between both species (Figure 1A). At 25 °C, the highest growth rates also corresponded to T. harzianum at 0.995 and 0.98 αw, whereas at 0.95 and 0.90 αw, F. sudanense showed higher values than T. harzianum (Figure 1B).

Figure 1.

Figure 1

Open in a new tab

Influence of water activity (αw) and temperature on the growth rates of F. sudanense in dual culture with T. harzianum. A: 15 °C, B: 25 °C.

Macroscopic Study of the Interaction

Figures 2 and 3 show the interactions between F. sudanense and T. harzianum on the dual culture on PDA adjusted at different αw values and incubated at two temperatures after four weeks. Initially, both fungi were grown until they came into contact. Later, T. harzianum inhibited F. sudanense growth at 0.995, 0.98, and 0.95 αw at 25 °C and at 0.995 and 0.98 at 15 °C. T. harzianum grew and surrounded the F. sudanense colony at 0.995 and 0.98 αw at both temperatures (Figures 2 and 3). T. harzianum overgrew the F. sudanense colony at 0.995, 0.98, and 0.95 αw at 25 °C (Figure 3). At 15 °C and 0.995 αw, T. harzianum also overgrew and surrounded the F. sudanense colony, while at this temperature and 0.98 αw, T. harzianum showed incipient overgrowth of the F. sudanense colony. Interestingly, F. sudanense produced an intense violet pigment in media under all the evaluated conditions (Figure 2).

Figure 2.

Figure 2

Open in a new tab

Dual cultures of F. sudanense and T. harzianum under 0.95, 0.98, and 0.995 αw at 15 °C. (a) Original plates and (b) enlarged images. T. harzianum did not grow at 0.90 αw.

Figure 3.

Figure 3

Open in a new tab

Dual cultures of F. sudanense and T. harzianum under 0.95, 0.98, and 0.995 αw at 25 °C. (a) Original plates and (b) enlarged images. T. harzianum did not grow at 0.90 αw.

Table 2 shows different interaction types and the ID between F. sudanense and T. harzianum in dual cultures at different water activities and temperatures after four weeks of incubation.T. harzianum antagonized F. sudanense at 0.995, 0.98, and 0.95 αw and 25 °C, whereas it dominated at 0.995 and 0.98 αw and 15 °C. As shown in Table 2, dominance upon contact was the main interaction observed on the dual cultures. The interaction “D” type (4/0) was assigned because T. harzianum inhibited F. sudanense colonies and, as mentioned above, this fungus overgrew the F. sudanense colonies after four weeks of incubation, except at 0.95 αw and 15 °C. The competitive ability of T. harzianum diminished as the water activity and temperature decreased. Under these conditions, both fungi grew slowly, and interaction “A” was assigned.

Table 2. Index of Dominance (ID) and Types of Interaction between F. sudanense and T. harzianum in Dual Cultures at Different Conditions after Four Weeks of Incubation.

    αwa
  
temperatures species 0.995 0.98 0.95 0.90c IDb
25 °C F. sudanense 0 0 0   0
T. harzianum 4 4 4   12
type of interaction   D D D    
15 °C F. sudanense 0 0 2   2
T. harzianum 4 4 2   10
type of interaction   D D B    
Open in a new tab
a

αw: Water activities.

b

ID: index of dominance according Magan and Lacey (1984).36 Values were obtained by adding the values assigned to each species according to the interaction with the different water activities. Types of interaction and corresponding values: A - mutual intermingling (1), B - mutual antagonism on contact or with free space between fungus colonies <2 mm (2), C - mutual antagonism at a distance (3), D - dominance on contact (4 for the dominant species, 0 for the inhibited species), and E - dominance at a distance (5 for the dominant species, 0 for the inhibited species).

c

Interaction discarded because there was no growth.

The numerical values assigned to each fungus (ID) showed that T. harzianum was the species that dominated F. sudanense except at the lowest αw. The ID indicated the antagonistic potential of T. harzianum against F. sudanense. This index clearly assessed this fungus’s ability to reduce F. sudanense growth under different water activity and temperature conditions except at 0.95 αw and 15 °C, and 0.90 αw.

Microscopic Analysis of the Interaction

The microscopic observations from the interaction zone revealed initial contact between both fungi and the attachment of T. harzianum to the F. sudanense hyphae. Penetration with or without the formation of appressorium-like structures was also observed (Figure 4A–F). Tight attachment, conidia concentration, and hyphae woven or intertwined into a compact mass with strong hyphal compression were easily observed from the interaction zones section under light and cryo-scanning electron microscopy (Figure 4G–I). The presence of F. sudanense hyphae with a wrinkled or shrunken appearance, compared to the hyphae grown alone, was observed (Figure 5B,D) (plasmolysis).

Figure 4.

Figure 4

Open in a new tab

Micrographs of the interaction between F. sudanense and T. harzianum. (A, D, G) Light micrographs and (B, C, E, F, H, I) cryo-scanning electron micrographs showing the initial contact (A, B) and contact and penetration (C, D, E, F) with or without the formation of appressorium-like structures (E). conidia concentration of F. sudanense (G) and hyphae woven and intertwined into a compact mass with a strong hyphal compression (H, I). The arrow indicates the contact and penetration zones.

Figure 5.

Figure 5

Open in a new tab

Cryo-scanning micrographs of different types of mycoparasitism used by T. harzianum against F. sudanense. (A, C) Coiling, (B, D) plasmolysis, and (E, F) veil formation wrapping the false heads and microconidia of F. sudanense.

The F. sudanense hyphae that were coiled by the T. harzianum hyphae were observed (Figure 4A,C). Interestingly, a veil-like formation that wrapped false heads and microconidia of F. sudanense was observed (Figure 4E,F). Thus, T. harzianum antagonized F. sudanense with initial contact by coiling and hyphae plasmolysis. Clear evidence for direct mycoparasitism was herein recorded.

Discussion

Growth of F. sudanense and T. harzianum was significantly affected by water activity and temperature in PDA medium. These results indicated that, when αw and temperature values were high, both fungi rapidly developed, whereas growth was slower under nonoptimal conditions (αw and temperature values were low), or no growth was detected after six days.

The optimal conditions for F. sudanense growth were similar to those previously described for others Fusarium spp., such as F. oxysporum f. sp. lycopersici, F. sambucinum, F. verticilliodes, and F. proliferatum.(24−26) In addition, different authors have demonstrated the influence of abiotic factors, such as water activity and temperature, on the ability of different Fusarium species to germinate, grow, and produce mycotoxins.27,28

Our results indicated that T. harzianum CECT 20736 suppressed F. sudanense growth at 0.995 and 0.98 αw at 15 and 25 °C. In particular, the growth rates for T. harzianum CECT 20736 evaluated corroborate previous observations made by Sempere and Santamarina22,26 with the same strain and agree with those reported by Santamarina and Roselló29 using T. harzianum LBVB 1010.

According to the results obtained in dual culture assay and our macroscopic study, both fungal species required a high moisture content to achieve their optimal growth. Moreover, the highest T. harzianum growth rates versus those of F. sudanense allowed us to suggest that this strain offers a very high potential as a biocontrol agent against F. sudanense by using competition for space or resources as its main mechanisms. Our microscopic studies also detected that T. harzianum used direct mechanisms (mycoparasitism) to control F. sudanense, such as penetration, plasmolysis, and coiling among others. These results suggest that the T. harzianum strain evaluated in this study could release cellular enzymes capable of penetrating and degrading cell walls and causing plasmolysis, such as chitinase, protease, and glucanase enzymes. Several authors have demonstrated that competition for space or resources and mycoparasitism are the main mechanisms used by different Trichoderma species to carry out the biocontrol of fungal phytopathogens.5,17−19,30−34 The fungal ability to occupy certain ecological niches in nature depends on their capacity to compete against other microorganisms and environmental conditions to successfully develop among other factors.35 The cited authors recognized a range of interspecific fungal interactions that are influenced by environmental conditions. We herein used the method proposed by Magan and Lacey36 to determine the type of interaction and the ID that would indicate the ability of a fungus to dominate under different in vitro conditions. Determining in advance how the species behave upon interacting and under different environmental conditions (water availability and temperature) is important since it allows us to know which species will dominate in a specific ecosystem.

Different Fusarium species are known to cause latent infections, which are considered one of the highest parasitism levels.37 Verhoeff38 stated that true latent infection must involve a parasitic relation that eventually induces symptoms. In agreement with what Sinclair states,37 knowledge of fungal-causing latent infections contributes to developing more effective disease management measures. Interestingly, we isolated and identified F. sudanense from asymptomatic wheat grains of different samples, which causes seedling blight and seed rot.4 Thus, we suggest that it could be a latent pathogen awaiting specific environmental conditions to switch to a pathogenic state. Consequently, its early detection in grains could avoid disease and loss of wheat crops. It is important to highlight that the environmental conditions for optimal F. sudanense growth obtained herein (high humidity and temperate temperatures) and in another study by the same authors4 occur in the typical agro-ecological wheat region of Argentina until stem elongation, which is growth stage 30 according to Zadoks et al.39 As T. harzianum also grew under these same conditions, we suggest that both fungi can interact and, consequently, T. harzianum can dominate F. sudanense in field scenarios. These findings are of much importance today because biological control is an environmentally friendly and safer tool to be considered among current integrated disease management strategies applied to wheat crops.

Conclusions

In conclusion, the results herein presented showed that the T. harzianum strain studied had successful biocontrol activity and ability to compete against F. sudanense. Competition for space and nutrients and direct mycoparasitism were the mechanisms used by T. harzianum for the biological control of F. sudanense. The initial contact between both fungi and the attachment of T. harzianum to the F. sudanense hyphae, its penetration with or without the formation of appressorium-like structures, coiling, mycelial plasmolysis, and a clearly demarcated veil formation were all recorded for the first time.

According to our results, by demonstrating the ability and aggressive competitive nature of T. harzianum, we could protect wheat seedlings against F. sudanense when applied to seed or soil treatments.

Materials and Methods

Fungal Strains

The F. sudanense strain was obtained from asymptomatic wheat grains of the cultivar Klein Yarará at the Laboratory of the CIDEFI, FCAyF, UNLP, Buenos Aires province (Argentina), and incorporated into the Colección Española de Cultivos Tipo (CECT) of Universidad de Valencia (UV, Spain), accession no. CECT 20938; GenBank accession nos. MF540540, MF581918, and MH411162.

The T. harzianum strain (CECT 20736) was provided by the CECT (UV), and it was previously obtained from corn grains from samples of Valencia, Spain. The fungus was identified by the Centraalbureau voor Schimmelcultures (CBS), Fungal Biodiversity Centre and included in the Banco de Datos de Biodiversidad de la Comunidad Valenciana (BDBCV-General: 659374) (https://www.gbif.org/occurrence/1501023929).

Both strains were maintained on potato dextrose agar (PDA) at the Laboratorio de Botánica, Escuela Técnica Superior de Ingeniería Agronómica y del Medio Natural (ETSIANM) of UPV, Spain, until used.

Interaction between F. sudanense and T. harzianum

To evaluate the antifungal activity of T. harzianum against F. sudanense at different water activities and temperatures, both fungi were kept on 2% PDA and incubated at 25 °C in the dark for seven and five days, respectively. In order to prepare culture media with different water availabilities, PDA (pH 5.5) was used as a basic medium, which was adjusted by adding glycerol and distilled water following the technique by Sempere and Santamarina.40 The prepared water activities (αw) were 0.90, 0.95, 0.98, and 0.995. The dual culture technique was used to screen the potential biocontrol of T. harzianum against F. sudanense. Mycelial disks (8 mm diameter) of each fungus obtained from the edge of the colonies developed on 2% PDA were placed on the opposite side (45 mm apart) in 90 mm Petri dishes containing PDA adjusted to different αw values. The controls were performed by placing only disks of F. sudanense and T. harzianum on all the adjusted media. Plates with the same water activity were placed inside hermetic plastic boxes containing a glycerol water solution with an equilibrium relative humidity value identical to the αw of the plates.40 Then, they were incubated at 15 and 25 °C in darkness. Treatments were performed by combining the four water activities and incubating at both temperatures. Each treatment was replicated four times. Fungal F. sudanense and T. harzianum growth was examined at 24 h intervals for six days, and the diameter of the growing colony was measured in two directions at right angles to each other. A linear regression of the increase in radius (in mm) against time (in days) was used to obtain the growth rates (mm/day) for each set of treatments using the computer software Microsoft Excel. An analysis of variance (ANOVA) of the growth rates obtained was performed using GenStat Release 12.1 (2009) where the factors were species, water activities, and temperatures. Means were compared by an LSD test at P ≤ 0.05.

Macroscopic Study of the Interaction

After obtaining the growth rates, Petri plates were maintained for 4 weeks at 15 and 25 °C to analyze the macroscopic evolution of the fungal species growing in the interaction. At this time, the index of dominance (ID) of each fungus, which indicates the ability of fungi to compete or dominate under the prescribed environmental conditions, was determined. Previously, numerical values were given to both fungi to obtain the ID. With these values, interaction types were determined following Magan and Lacey:36 mutual intermingling (1), mutual antagonism on contact or with a free space among fungus colonies <2 mm (2), mutual antagonism at a distance (3), dominance upon contact (4 for the dominant species, 0 for the inhibited species), and dominance at a distance (5 for the dominant species, 0 for the inhibited species). The results were presented for each αw and temperature.

Microscopy Study of the Interaction

In order to elucidate the mechanisms of antagonism used by T. harzianum against F. sudanense under in vitro conditions, a dual microculture technique was used. Both fungal species were inoculated 5 mm apart on PDA squares adjusted at 0.995 αw (99% equilibrium relative humidity), mounted on a glass rod inside 90 mm Petri plates under sterile conditions. Coverslips were placed on the agar. The αw level was maintained by placing filter paper disks impregnated with a solution at 0.995 αw and then were aseptically placed on the Petri dishes. Five plates with dual microcultures were incubated at 25 °C for 3–5 days until observation. The controls were the plates with each fungus alone. A microscopic study was performed with the samples of individual fungi and the samples from the interaction zone from the dual microcultures. Examinations were made under a light microscope (Olympus PM-10AK3) at the Laboratorio de Botánica of the ETSIANM of the UPV, Spain.

The cryo-scanning electron analysis was undertaken using dual microculture but no coverslips were placed on the PDA adjusted at 0.995 αw. Samples of the interaction zone F. sudanense–T. harzianum were mounted on a stub and frozen on liquid nitrogen. Then, samples were sublimed at −90 °C for 15 min, covered with gold for 30 s according the technique used by Sempere and Santamarina.41 A scanning electron microscope JEOL JSM 5410 was used at the UPV Electronic Microscopic Service (Spain).

Acknowledgments

This work was supported by the Escuela Técnica Superior de Ingeniería Agronómica y del Medio Natural (ETSIANM), UPV, Spain, and by Facultad de Ciencias Agrarias y Forestales of the Universidad Nacional de La Plata (grant no. 11A 296), Argentina.

The authors declare no competing financial interest.

References

  1. Arnold A. E. Understanding the diversity of foliar endophytic fungi: progress challenges, and frontiers. Fungal Biol. Rev. 2007, 21, 51–66. 10.1016/j.fbr.2007.05.003. [DOI] [Google Scholar]
  2. Porras-Alfaro A.; Bayman P. Hidden fungi, emergent properties: endophytes and microbiomes. Annu. Rev. Phytopatol. 2011, 49, 291–315. 10.1146/annurev-phyto-080508-081831. [DOI] [PubMed] [Google Scholar]
  3. Keswani C.; Singh H. B.; Hermosa R.; et al. Antimicrobial secondary metabolites from agriculturally important fungi as next biocontrol agents. Appl. Microbiol. Biotechnol. 2019, 103, 9287–9303. 10.1007/s00253-019-10209-2. [DOI] [PubMed] [Google Scholar]
  4. Vanegas A. M. M.; Osorno J. C.; Marín A. Metabolitos secundarios en Trichoderma spp. y sus aplicaciones biotecnológicas agrícolas. Actu. Biol. 2019, 41, 32–44. 10.17533/udea.acbi.v41n111a02. [DOI] [Google Scholar]
  5. Moussa T. A. A.; Al-Zahrani H. S.; Kadasa N. M. S.; Ahmed S. A.; de Hoog G. S.; Al-Hatmi A. M. S. Two new species of the Fusarium fujikuroi species complex isolated from the natural environment. Antonie van Leeuwenhoek 2017, 110, 819–832. 10.1007/s10482-017-0855-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Larran S.; Santamarina Siurana M. P.; Caselles J. R.; Simón M. R.; Perelló A. Fusarium sudanense, endophytic fungus causing typical symptoms of seedling blight and seed rot on wheat. J. King Saud Univ. Scie. 2018, 32, 468–474. 10.1016/j.jksus.2018.07.005. [DOI] [Google Scholar]
  7. Larran S.Estudio de la micobiota endofítica asociada al trigo y soja y su significancia en la interacción con patógenos fúngicos;. Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata. 2016http://sedici.unlp.edu.ar/handle/10915/54499.
  8. Shi W.; Tan Y.; Wang S.; Gardiner D.; De Saeger S.; Liao Y.; Wang C.; Fan Y.; Wang Z.; Wu A. Mycotoxigenic potentials of Fusarium species in various culture matrices revealed by mycotoxin profiling. Toxins 2017, 9, 6. 10.3390/toxins9010006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bockus W.W.Compendium of wheat disease and pests; 3rd edn., American Phytopathology Society: St. Paul, 2010. [Google Scholar]
  10. Kuldau G.A.; Yates I. E.. Evidences for Fusarium endophytes in cultivated and wilt plants. InMicrobial endophytes; Bacon C. W.; Bacon J. F. Jr. Eds; Dekker: New York, 2000; pp. 85–117. [Google Scholar]
  11. Leslie J.F.; Summerell B.A. eds. The Fusarium Laboratory Manual: Blackwell Publishing: USA. 2006. [Google Scholar]
  12. Perincherry L.; Lalak-Kańczugowska J.; Stępień Ł. Fusarium-Produced Mycotoxins in Plant-Pathogen Interactions. Toxins 2019, 11, 664. 10.3390/toxins11110664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Edwards J.; Auer D.; de Alwis S.-K.; Summerell B.; Aoki T.; Proctor R. H.; Busman M.; O’Donnell K. Fusarium agapanthi sp. nov., a novel bikaverin and fusarubin-producing leaf and stem spot pathogen of Agapanthus praecox (African lily) from Australia and Italy. Mycologia 2016, 108, 981–992. 10.3852/15-333. [DOI] [PubMed] [Google Scholar]
  14. Andrade J. F.; Satorre E. H. Single and double crop systems in the Argentine Pampas: Environmental determinants of annual grain yield. Field Crop Res. 2015, 177, 137–147. 10.1016/j.fcr.2015.03.008. [DOI] [Google Scholar]
  15. Ortega L. M.; Dinolfo M. I.; Astoreca A. L.; Alberione E. J.; Stenglein S. A.; et al. Molecular and mycotoxin characterization of Fusarium graminearum isolates obtained from wheat at a single field in Argentina. Mycol. Prog. 2016, 15, 1. 10.1007/s11557-015-1147-7. [DOI] [Google Scholar]
  16. Perelló A. E.; Dal Bello G. M. Suppression of tan spot and plant growth promotion of wheat by synthetic and biologic inducers in field conditions. Ann. Appl. Biol. 2011, 158, 267–274. 10.1111/j.1744-7348.2011.00460.x. [DOI] [Google Scholar]
  17. Monte E. Understanding Trichoderma: Between biotechnology and microbial ecology. Int. Microbiol. 2001, 4, 1–4. [DOI] [PubMed] [Google Scholar]
  18. Harman G. E.; Petzoldt R.; Comis A.; Chen J. Interactions between Trichoderma harzianum strain T22 and maize Inbred line Mo17 and effects of these interactions on diseases caused by Pythium ultimum and Colletotrichum graminicola. Phytopathology 2004, 94, 147–153. 10.1094/PHYTO.2004.94.2.147. [DOI] [PubMed] [Google Scholar]
  19. Stocco M.C.Control biológico de Mycosphaerella graminicola, patógeno del trigo, con cepas de Trichoderma harzianum caracterizadas por su morfología, fisiología, actividad enzimática y molecular. Tesis doctoral. Facultad de Ciencias Naturales y Museo; Universidad Nacional de La Plata: 2014. [Google Scholar]
  20. Waghunde R. R.; Shelake R. M.; Sabalpara A. N. Trichoderma: A significant fungus for agriculture and environment. Afr. J. Agric. Res. 2016, 11, 1952–1965. [Google Scholar]
  21. Perelló A.; Lampugnani G.; Abramoff C.; Slusarenko A.; Dal Bello G. Suppression of seed-borne Alternaria arborescens and growth enhancement of wheat with biorational fungicides. Inter. J. Pest Manage. 2017, 63, 157–165. 10.1080/09670874.2016.1252478. [DOI] [Google Scholar]
  22. Sempere F.; Santamarina M. P. Antagonistic interactions between fungal rice pathogen Fusarium verticillioides (Sacc.) Nirenberg and Trichoderma harzianum Rifai. Annals of Microbiology 2009, 59, 259–266. 10.1007/BF03178326. [DOI] [Google Scholar]
  23. Lacey J.; Magan N.. Fungi in cereal grain: their occurrence and water and temperature relationship. In Cereal grain: Micotoxins, fungi and quality in drying and storage; Chelcowsky J. Ed. Elsevier: Amsterdam; 1991; pp. 77–118. [Google Scholar]
  24. RosellóCaselles J. L.; Sempere F.; Santamarina M. P. Respuestas al agua y a la temperatura del agente causante de la fusariosis del tomate. Fusarium oxysporum f.sp. lycopersici. Phytoma 2004, 162, 98. [Google Scholar]
  25. Samapundo S.; Devliehgere F.; de Meulenaer B.; Debevere J. Effect of water activity and temperature on growth and the relationship between fumonisin production and the radial growth of Fusarium verticillioides and Fusarium proliferatum on corn. J. Food Prot. 2005, 68, 1054–1059. 10.4315/0362-028X-68.5.1054. [DOI] [PubMed] [Google Scholar]
  26. Ferre F. S.; Santamarina M. P. Efficacy of Trichoderma harzianum in suppression of Fusarium culmorum. Ann. Microbiol. 2010, 60, 335–340. 10.1007/s13213-010-0047-y. [DOI] [Google Scholar]
  27. Llorens A.; Mateo R.; Hinojo M. J.; Valle-Algarra F. M.; Jiménez M. Influence of environmental factors on the biosynthesis of type B trichothecenes by isolates of Fusarium spp. From Spanish crops. Int. J. Food Microbiol. 2004, 94, 43–54. 10.1016/j.ijfoodmicro.2003.12.017. [DOI] [PubMed] [Google Scholar]
  28. Magan N.; Medina A. Integrating gene expression, ecology and mycotoxin production by Fusarium and Aspergillus species in relation to interacting environmental factor. World Mycotoxin J. 2016, 9, 673–684. 10.3920/WMJ2016.2076. [DOI] [Google Scholar]
  29. Santamarina M. P.; Roselló J. Influence of temperature and water activity on the antagonism of Trichoderma harzianum to Verticillium and Rhizoctonia. Crop Prot. 2006, 25, 1130–1134. 10.1016/j.cropro.2006.02.006. [DOI] [Google Scholar]
  30. Prasad R. D.; Rangeshwaran R.; Hegde S. V.; Anuroop C. P. Effect of soil and seed application of Trichoderma harzianum on pigeonpea wilt caused by Fusarium udum under field conditions. Crop Prot. 2002, 21, 293–297. 10.1016/S0261-2194(01)00100-4. [DOI] [Google Scholar]
  31. Perello A. E.; Monaco C. I.; Moreno M. V.; Cordo C. A.; Simon M. R. The effect of Trichoderma harzianum and Trichoderma koningii on the control of tan spot (Pyrenophora tritici-repentis) and leaf blotch (Mycosphaerella graminicola) of wheat under field conditions in Argentina. Biocont. Scie. Tech. 2006, 16, 803–813. 10.1080/09583150600700099. [DOI] [Google Scholar]
  32. Dal Bello G.; Mónaco C.. Manejo de enfermedades con agentes biocontroladores. InEnfermedades del trigo; Cordo C.; Sisterna M. Eds.; Edulp: La Plata, Buenos Aires; 2014; pp. 401–420. [Google Scholar]
  33. Larran S.; Simón M. R.; Moreno M. V.; Santamarina Siurana M. P.; Perelló A. Endophytes from wheat as biocontrol agents against tan spot disease. Biol. Control 2016, 92, 17–23. 10.1016/j.biocontrol.2015.09.002. [DOI] [Google Scholar]
  34. Nakkeran S.; Renukadevi P.; Aiyanathan K. E. A.. Exploring the potential of Trichoderma for the management of seed and Soil-Borne diseases of crops. InIntegrated pest management of tropical vegetable crops; Muniappan R.; Heinrichs E. A. Eds.; Springer: USA, 2016; pp. 77–13. [Google Scholar]
  35. Magan N.; Aldred D.. Environmental fluxes and fungal interactions: maintaining a competitive edge. InStress in yeasts and filamentous fungi, Avery S.V.; Stratfford M.; van West P. Eds.; Elsevier: Amsterdam, 2007; pp. 19–35. [Google Scholar]
  36. Magan N.; Lacey J. The effect of water activity, temperature and substrate on interactions between field and storage fungi. Trans. Br. Mycol. Soc. 1984, 82, 83–93. 10.1016/S0007-1536(84)80214-4. [DOI] [Google Scholar]
  37. Sinclair J. B. Latent infection of soybean plants and seeds by fungi. Plant Dis. 1991, 75, 220–224. 10.1094/PD-75-0220. [DOI] [Google Scholar]
  38. Verhoeff K. Latent infections by fungi. Annu. Rev. Phytopathol. 1974, 12, 99–110. 10.1146/annurev.py.12.090174.000531. [DOI] [Google Scholar]
  39. Zadoks J. C.; Chang T. T.; Konsak C. F. A. A decimal code for the growth stages of cereals. Eucarpia Bulletin 1974, 7, 415. [Google Scholar]
  40. Sempere F.; Santamarina M. P. Ecofisiología de Drechslera oryzae Subram. & Jain en condiciones in vitro. Phytoma 2006, 178, 49–50. [Google Scholar]
  41. Sempere F.; Santamarina M. P. Cryo-scanning electron microscopy and light microscopy for the study of fungi interactions. Microsc. Res. Tech. 2011, 74, 207–211. 10.1002/jemt.20893. [DOI] [PubMed] [Google Scholar]

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES