Diseases caused by fungal, oomycete, bacterial, and viral pathogens produce severe reductions in yield and quality of crop harvests, which in many cases lead to important economic losses for farmers (Vidhyasekaran, 2004). Therefore, to combat pathogens and reduce infestations, farmers apply a multitude of different pesticides that in many cases have a negative effect on the environment and human/animal health. Moreover, as a result of the extensive usage of chemicals in disease crop management, development of resistance to modern fungicides in field populations of fungal and oomycete pathogens have increased (Fernández-Ortuño et al., 2015; Saville et al., 2015).

Due to the described problems much effort is concentrated on finding new efficient, economical, low environmental-impact options to control pathogens. One of the most promising approaches is the use of plant defense elicitors, alone or in combination with pesticides or beneficial microorganisms (Walters et al., 2013). Elicitors or plant defense activators normally induce an incomplete, broad range, systemic resistance (Walters and Fountaine, 2009) as displayed by a significant reduction in disease symptoms caused by different types of pathogens (Kuć, 2006).

Over the last two decades, several resistance elicitors have been described, and they have been classified into two main groups, molecules that are, or mimic, phytohormones or compounds that resemble the presence of a plant enemy (Heil, 2014). Therefore, pathogen protection in plants can be induced by the exogenous application of phytohormones that activate systemic resistance responses such as salicylic acid (SA) and jasmonic acid (JA) (Pieterse et al., 2009). In addition, some volatile derivatives or artificial compounds that structurally resemble these hormones and cause similar plant responses have been characterized, such as 2,6-dichloroisonicotinic acid (INA) or benzothiadiazole (BTH), also known as acibenzolar-S-methyl (ASM). In the second group, resistance can be induced by the application of: (i) molecules that indicate the presence of pathogens or microbes and thus are perceived by plants as ‘pathogen-associated molecular patterns’ (PAMPs) or ‘microbe associated molecular patterns’ (MAMPs) and of (ii) plant extracts containing break-down products known as ‘damage-associated molecular patterns’ (DAMPs). PAMPs- or MAMPs-like molecules can form part of the cell wall of microorganisms, be commonly secreted by them or represent other invariable characteristics of microbial surfaces (Heil, 2014). In addition to the two aforementioned groups of elicitors, a third type of compounds including mineral nutrients such as phosphorus (P), potassium (K), and silicon (Si), fertilizers containing phosphite (phosphorous acid) and the non-protein amino acid DL-3-amino-n-butanoic acid (BABA) have been shown to induce pathogen defense responses and enhance plant disease protection (Reglinski et al., 2014).

Many studies have demonstrated the successful use of different elicitors for suppression of diseases in a wide variety of crops (Lyon et al., 1995; Aziz et al., 2006; Elmer and Reglinski, 2006; Renard-Merlier et al., 2007; French-Monar et al., 2010). The first disease resistance activator, probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide), was registered in Japan more than 30 years ago as Oryzemate to control rice blast (Iwata et al., 2004). Since then, many other chemical and biological activators have been commercially developed and registered as agricultural products including BION or Actigard (formulated with BTH; Syngenta, Switzerland), Milsana (Reynoutria sachalinensis extract; KHH BioScience, United States), Elexa (chitosan; SafeScience, United States; and Glycogenesys Inc., United States) and Messenger or ProAct (harpin protein; Plant Health Care, United States) also commercialized as Employ (H&T Health Promoter, United States) (Walters et al., 2013). Currently, commercial formulations based on different PAMP/MAMP compounds such as flagellin, elicitins, harpins, cerebrosides, rhamnolipids, Sm1 protein and yeast derived-elicitors, are being developed (Elmer and Reglinski, 2006; Iriti et al., 2011; Dafermos et al., 2012).

Switching on the plant innate immunity response through treatment with different PAMP/MAMP molecules has demonstrated the effectiveness of this technology to control diseases caused by virus, bacteria, oomycetes and a wide range of biotrophic, hemibiotrophic, and necrotrophic fungi in many different plant species (Iriti et al., 2011; Li et al., 2011; Choi et al., 2012; Dafermos et al., 2012; Sanchez et al., 2012; Quang et al., 2015). The use of induced resistance to improve plant protection against pathogens has mostly been implemented in intensive and greenhouse grown crops such as fruits, vegetables, and ornamentals, although there are a considerable number of examples of successful application in extensive crop production systems including maize, wheat, and barley (Vallad and Goodman, 2004; Reglinski et al., 2014). In addition to trigger protection against plant pathogens, some elicitors have also been shown to affect physiological responses in plants, resulting in an increased crop yield and quality through improved nutrient assimilation or increased tolerance to low or high temperatures and water abiotic stress (Khan et al., 2009; Calvo et al., 2014; Chuang et al., 2014).

Traditionally, defense elicitor-based products has been categorized as plant strengtheners or biopesticides but has recently been included in the more broad term biostimulant, defined as “a formulated product of biological origin that improves plant productivity as a consequence of the novel or emergent properties of the complex of constituents and not as a sole consequence of the presence of known essential plant nutrients, plant growth regulators, or plant protective compounds” (Yakhin et al., 2017). The main reason behind this new definition is to stimulate the development of harmless products of natural origin in agriculture production and facilitate regulation and registration governing these compounds (biostimulants).

We have previously described the isolation and characterization of AsES, an extracellular subtilisin-like protease produced by the opportunistic strawberry pathogen Acremonium strictum strain SS71 (Castagnaro et al., 2012; Chalfoun et al., 2013). This 34-kDa fungal protein has previously been shown to induce an innate defense response and generate protection against anthracnose in strawberry (Chalfoun et al., 2013; Hael-Conrad et al., 2018) and gray mold in Arabidopsis under controlled growing conditions (Hael-Conrad et al., 2015).

The important disease protection induced by AsES demonstrated in two different plant species and against different pathogens stimulated us to try and develop a novel broad-range crop disease control product, PSP1 (acronym for Plant Stimulation and Protection). In this study, we show the technological development of PSP1 from initial production and subsequent formulation, including tests of stability, quality control development, toxicological studies, adjustments of optimal concentration and application timing for disease control and testing of broad-range protection against diseases in three economically important crop species in Argentina, target spot caused by the fungus Corynespora cassiicola (Berk and M. A. Curtis) C. T. Wei in soybean (Ploper, 2011), red stripe (Acidovorax avenae) in sugarcane (Fontana et al., 2013), and head blight (Fusarium graminearum Schwabe) in wheat (Martinez et al., 2014).

In this work, we have described the sequential development of a possible new biostimulant, PSP1, based on an elicitor of natural origin for sustainable disease control in several crop species of importance in Argentina. The technological development of PSP1 followed the steps described for many commercial agricultural products after initial characterization and patenting (Castagnaro et al., 2012), including: (i) the development of a low cost production and downstream-processing system, (ii) formulation for stable storage over longer periods, (iii) toxicology testing on non-target organisms, (iv) a simple handling and application method, (v) criteria of quality control and finally (vi) product registration (Jeyarajan and Nakkeeran, 2000).

Considering that the subtilisin-like protease AsES is the only defense-inducing principle identified until now in the extracellular growth medium of A. strictum SS71, fungal growth conditions that stimulated extracellular protein secretion and protease activity were defined as the most important criteria when developing the fermentation process. Previously we have produced AsES by statically growing A. strictum using PDB as a substrate (Chalfoun et al., 2013) but considering that 30–40% of the production cost of industrial enzymes is estimated to be accounted for by the cost of the growth medium a cheaper alternative (3–5 times), SMB, was tested. Soybean meal is recognized as a potentially useful and cost-effective medium ingredient, because it is largely produced as a by-product during oil extraction (Gattinger et al., 1990) and have been successfully used to promote production of extracellular proteases by filamentous fungi (Agrawal et al., 2005; Haq and Mukhtar, 2007; Savitha et al., 2011). By employing SMB not only did we manage to significantly lower production costs but extracellular protein concentrations increased as well. Furthermore, when scaling up production fermentation times were significantly reduced by more than half demonstrating the usefulness of SMB as substrate for large-scale production of PSP1.

To control residual fungal growth and microbe contamination an acidification step of the supernatant was included in the production process. AsES is an alkaline protease and lowering the pH by acidification of the supernatant to pH 5.5 reduced its activity by 75% (Supplementary Figure 1A) but did not significantly affect its elicitor activity. Thermal variation had very little influence on the protease activity of PSP1 as it retained >90% of its activity when kept at 37°C, 45°C or 60°C at pH 7.5 for 30 min (Supplementary Figure 1B). Interestingly, purified AsES completely lacked enzymatic activity at temperatures above 70°C, whereas PSP1 exhibited some activity even at a temperature as high as 80°C (Supplementary Figure 1C). If this effect is due to enhanced stability of the protein by other constituents in the supernatant or increased instability of the purified protein or a combination of both factors remain to be elucidated. The preservation of enzymatic activity of PSP1 at relatively high temperatures (Supplementary Figure 1C) is in agreement with the observed stability of production batches at temperatures of 37–45°C (Table 3).

As previously pointed out we found that a high total extracellular protein concentration of PSP1 was not a perfect indicator of plant disease protection capacity. Therefore, as a previous study had demonstrated that the defense-inducing capability of AsES seems to be directly correlated to the protease activity of this protein (Chalfoun et al., 2013), a measurement of protease activity of PSP1 was tested as a rapid and reproducible quality control method to verify defense-inducing capacity. As all fermentation batches showing detectable protease activity were capable of inducing disease protection and the protease activity has been incorporated as a routine assay of batch quality control. It is however, important to notice that the exact role for this protease activity in plant defense induction is not known and further studies is therefore needed to elucidate the mechanism and possible targets for AsES.

Studies have shown that the concentration of an elicitor can determine the efficacy of the treatment in controlling diseases in different plant species. For example, high concentrations of harpin demonstrated efficient control of Penicillium expansum in apple (de Capdeville et al., 2002), whereas lower concentrations were more effective against rice bacterial blight (Chen et al., 2008). In this study we did not find a clear dose–response effect to PSP1-treatment and disease protection in neither strawberry nor soybean. Instead, it was observed that once a minimum protease activity had been reached a full protection was obtained, as increasing the protease activity, protection against strawberry anthracnose or STS did not increase. This threshold effect is in agreement with a previous study showing that the fungal supernatant derived from PDB-grown A. strictum culture diluted until 1:8 was able to induce full protection (equivalent to higher concentrations) against strawberry anthracnose but this effect was completely lost in a dilution 1:16 (Chalfoun, 2009). Nonetheless, this observation of a lack of dose-effect is somewhat contrasting to results obtained in Arabidopsis where an AsES dose–response was indeed observed in disease protection against the necrotrophic fungus Botrytis cinerea (Hael-Conrad et al., 2015). These differences could be explained by different challenging pathogens-plant systems, and differences between effects of PSP1 and purified AsES.

The sine qua non mandatory requirement for the registration of a biological product is the complete lack of animal toxicity and negative effects on the environment as they are intended to be used as tools in sustainable crop production and/or important components in IPM protocols or organic farming. Thus, the toxicity effect of PSP1 on non-target organisms such as insects, fish, mammals and birds was tested following IOBC standards for extended laboratory tests on natural substrates in accordance with guidelines of the National Food Safety and Quality Service (SENASA) for registration of a microbial biological control agent (ACBM), a microbial technical product (PTM) and a formulated microbial product (PMF) in Argentina. It is important to notice that there are no guidelines or even a category for registration in Argentina of compounds of natural origin to be used in agriculture practice, such as biostimulants. This fact has been pointed out to responsible authorities during the ongoing registration process of PSP1 and work has been initiated to include such products in the registration procedure in the future. Most importantly though, as demonstrated by these studies no adverse effects of this product are expected in field applications on the environment, animals or field workers and the PSP1 formulation should be a perfect complement in any type of sustainable crop disease management by itself or in combination with other products.

The development of biocontrol products based on plant defense elicitors such as MAMPs/PAMPs/DAMPs is a promising strategy for sustainable crop disease management, because they can render protection against a broad spectrum of plant pathogens in a wide-range of different plant species. However, elicitor molecules are specifically recognized in different host species and therefore induce different defense responses and so different protection level against diseases (Reboutier et al., 2007). For this reason it is important to thoroughly test defense induction and to evaluate potential disease control capacity in different crop species.

Soybean is by far the most important crop in Argentina and it was a logic step to test for disease protection capability of PSP1 in this species. The necrotrophic late season disease pathogen C. cassiicola, causing STS, which completely destroys the leaf cell as a strategy to obtain nutrients (Onesirosan et al., 1975; de Lamotte et al., 2007), was chosen for the initial IR studies. The occurrence of target spot on soybean has caused significant yield losses worldwide (Yorinori, 1997; Sinclair, 1999) and in recent years, due to the occurrence of C. cassiicola isolates resistant to fungicides, epidemics of the disease have been frequent in many soybean-growing regions in Brazil (Godoy et al., 2012; Teramoto et al., 2013) and Argentina (Ploper, 2011). No commercially relevant resistant cultivars are currently available and seed treatment or foliar spray with fungicides together with crop rotation are currently the most used disease control strategies (Almeida et al., 2005).

The time of application of an elicitor is a critical parameter in regard to its efficacy in controlling a plant disease. Generally elicitors/PAMPs are applied 2–5 days prior to a pathogen invasion (Qiu et al., 2001; de Capdeville et al., 2002, 2003). For example Messenger^® formulated with the bacterial protein harpin was effective to control blue mold disease in apple when applied 2 days before pathogen inoculation (de Capdeville et al., 2002) and the scab disease in rough lemon when applied 5 days before infection. However, the same product failed to control the disease when applied 10 days previous to pathogen inoculation (Agostini et al., 2003). In strawberry optimal anthracnose protection was obtained in plants pretreated with a supernatant of A. strictum 7 days before pathogen inoculation although good protection was also observed in plants treated 3 days previous to infection (Chalfoun, 2009). In accordance with these latter results PSP1-treatment of soybean plants was performed 3 and 7 days before C. cassiicola infection together with a 3 days post infection treatment. PSP1-treatment was effective against STS when applied 3 days before pathogen infection but this effect was markedly reduced when the time between PSP1-treatment and pathogen challenge increased to 7 days. Somewhat surprisingly a good protection was observed for plants treated with PSP1 3 days after infection with C. cassiicola although PSP1 do not possess any fungicide activity, indicating that PSP1-treatments 3 days before until 3 days after infection are effective against target spot and that the PSP1-induced response of the plant was able to combat the fungus in early stages of infection.

For soybean there are several reports demonstrating control of fungal diseases, although none of them have shown protection against C. cassiicola, by application of elicitor-based products under controlled growing conditions (Dann et al., 1998; Prapagdee et al., 2007; Nafie and Mazen, 2008; Lemes et al., 2011; Han et al., 2013; Cruz et al., 2014). However, there are only three cases in soybean, where elicitor-treatment have been described to be effective against necrotrophic pathogens; i.e., soil applications of mineral nutrient silicon (Si) reducing the incidence of frog-eye leaf spot and downy mildew diseases (Nolla et al., 2006), Regalia^®-treatment controlling Cercospora leaf blight caused by Cercospora kikuchii (Su et al., 2011), and EplT4, a peptide from Trichoderma asperellum T4, applied 12 h before infection protecting plants against Cercosporidium sofinum (Wang et al., 2013).

Once application dosage and treatment times for PSP1 had been optimized for target spot protection in soybean, a comparison of disease protection efficacy with the commercial elicitor BION was conducted. The active compound in BION is the SA-analog, BTH, which has been shown to be effective against a wide range of soybean pathogens causing damaging soil-borne diseases (Dann et al., 1998; Faessel et al., 2008; Nafie and Mazen, 2008; Abdel-Monaim et al., 2012; Sugano et al., 2012; Han et al., 2013). As demonstrated by our results plants treated with BION or PSP1 showed a similar protective effect against C. cassiicola in soybean and a very similar plant defense response as shown by accumulation of O2•− and H₂O₂ 4 h post-treatment and a strong induction of GmPR1 expression. The latter result demonstrated that both elicitors were able to activate the SA-mediated signaling pathway in this species, although a noticeable difference in GmPR1 induction kinetics was observed with an earlier and stronger gene induction in PSP1-treated plants as compared to BTH-treated ones (Lawton et al., 1996). This variation is probably dependent on the difference in SA-signaling, as previous studies have shown that treatment with AsES induces a transient accumulation of SA in strawberry (Chalfoun et al., 2013; Hael-Conrad et al., 2018) and Arabidopsis (Hael-Conrad et al., 2015), whereas BTH is acting downstream of SA accumulation (Friedrich et al., 1996). However, more studies on hormonal signaling pathways induced by AsES/PSP1 are needed to establish if there are any differences in the underlying molecular mechanisms of pathogen protection among different plant species.

The encouraging results from PSP1 application in soybean prompted us to test protection against diseases in an important crop species in the Northwest of Argentina. The reason behind selecting sugarcane and red stripe was twofold, first we wanted to test PSP1 in a monocot species and secondly to test if protection could be achieved against a bacterial disease as only fungal diseases had been tested previously. Although symptoms of red stripe in sugarcane were reported as early as 1895 in Argentina, the disease has not been considered a major problem until the last decade. The vastly increased number of incidents of this disease has been accompanying the implementation of new agricultural techniques in Argentina such as green-cane harvesting and crop rotation with soybean (Fontana et al., 2013). This situation has caused an increasing attention to the disease and because red stripe can significantly decrease sucrose recovery in susceptible varieties there is great concern how to reduce infection rates (Johnson et al., 2016). Actually, the only effective way for controlling red stripe in sugarcane has been the replacement of susceptible commercial varieties with more resistant ones (Martin et al., 1989) and it is therefore of great interest to develop alternative disease management strategies to be able to better control the disease in susceptible sugarcane varieties with good agronomical characteristics. The induced disease protection against red stripe in a susceptible sugarcane variety indicated that application of PSP1 is not only an interesting alternative for red stripe control in sugarcane but also suggested that PSP1 is a viable alternative for disease control in monocots. In addition, this is the first example of a successful protection against a bacterial disease mediated by AsES. These results taken together strongly indicates that AsES is acting as a PAMP/DAMP inducer and that the signaling is well conserved within the plant kingdom as has been seen for other proteinaceous inducers originating from microorganisms such as harpin and flagellin (or the peptide Flg22).

In parallel with testing PSP1 in sugarcane a 2-year field trial in wheat was conducted to test protection against F. graminearum, the principal causal agent of FHB in cereals. FHB is a devastating disease causing important losses in the commercial value of grains in both wheat and barley by reducing grain yields and by production of mycotoxins harmful to both humans and animals (Goswami and Kistler, 2004). Wheat is the most important winter cereal in Argentina with more than 5.000.000 ha planted and several FHB outbreaks have occurred during the last 60 years with estimated yield losses between 20 and 50% (Stenglein, 2009). The two main strategies to control this disease have been development of resistant plant varieties and application of broad range fungicides, but neither of these approaches have been very successful (Mesterhazy, 2003; Trail, 2009). Due to the lack of efficient disease management of this important disease, several studies on alternative biological treatments have been tested. An isolate of Pseudomonas fluorescens was reported to enhance resistance to Fusarium seedling blight and head blight caused by Fusarium culmorum in wheat by inducing both local and systemic responses (Khan et al., 2006; Khan and Doohan, 2009a,b). Further studies revealed that plant hormones indole acetic acid (IAA) and abscisic acid are involved in the P. fluorescens-mediated control of FHB in barley and that application of IAA to plants grown at mid-anthesis 24 h before pathogen infection, reduced FBH symptoms and increased yield (Petti et al., 2012). From these results it was suggested that IAA might offer a realistic treatment for the control of diseases such as FHB, where crops have a limited and clearly defined period of infection, mid-anthesis in the case of FHB (Petti et al., 2012). A significant protective effect against wheat FHB caused by F. graminearum has also been demonstrated for the known defense-inducer BABA (Zhang et al., 2007). Surprisingly, foliar application of BTH has widely been reported to reduce the severity of various fungal infections in wheat but it does not provide resistance to FHB (Yu and Muehlbauer, 2001). In addition to the above cited studies on successful application of compounds of natural origin for FHB management, results from the 2-year field trial studying diminished seed weight and viability caused by F. graminearum indicated that PSP1 provides an additional tool to effectively control this pathogen. The weight value of a thousand seeds constitutes a good parameter to measure yield loss caused by the inoculation with Fusarium spp. causing FHB and has previously been used by other authors (Petti et al., 2012). However, to confirm that this observed effect is reproducibly significant at total yield level, it is necessary to perform field trials determining total yields of plots under different growth conditions.

In this later study, we demonstrated that disease protection against F. graminearum by application of PSP1 was dependent on the plant genotype, since a greater impact was observed for the more susceptible genotype. This observation has previously been reported for other pathogen defense-inducing compounds including INA and BTH, which both showed better protection on more susceptible cultivars of soybean when challenged with Sclerotinia sclerotiorum (Pawlowski, 2015). The application of INA to soybean reduced natural infection of white mould by 20–70% in cultivars that are considered highly susceptible to this fungus, whereas the effect was much lower in resistant cultivars (Dann et al., 1998). However, effects of treatments in wheat has been more variable as application of JA resulted in an 85% decrease in Tilletia indica infection levels in susceptible varieties compared to 50% in resistant ones (Dutt et al., 2011), whereas Desmond et al. (2006) showed that MeJA treatment significantly delayed development of crown rot disease caused in wheat seedlings by the necrotrophic fungus Fusarium pseudograminearum in a genotype-independent manner.

From the combined disease protection studies in strawberry, soybean, sugarcane, and wheat it is obvious that although PSP1 is capable of inducing the plant defense against pathogens in all four species, it is necessary to optimize application procedure of PSP1 for maximum disease protection in the individual plant species.

It is important to observe that for all disease protection assays reported in this study, pathogens were artificially infested, and that all experiments except the wheat trial were performed under controlled growing conditions. This situation is very different from field growing conditions where plants are continuously exposed to pathogens and climate changes, constantly activating different biotic and abiotic defense responses as well as affecting plant hormones homeostasis. Another important factor to take into account is the possible energy costs generated by defense-inducing treatments against pathogens in the field, especially under poor nutrition conditions. For example, fitness costs of BTH-induced resistance in wheat were observed when nitrogen was limiting but undetectable in well fertilized plants (Heil et al., 2000). Thus the ability or requirement to respond to a pathogen attack may be reduced or unnecessary, but will be energy requiring and could therefore negatively affect yields, especially under poor nutrition conditions (Smedegaard-Petersen and Tolstrup, 1985).

It is therefore important to conduct multi-fold field trials under different climate conditions to be able to properly evaluate the protective effect of PSP1, to get an idea of their true value in crop disease management as there are several examples of defense activators that have performed well when used to control pathogen infections in laboratory or greenhouse conditions but have shown greater variability when applied in field environment (Hopkins, 2002; Agostini et al., 2003; Graham and Leite, 2004; Obradovic et al., 2005; Keinath et al., 2007; Walters and Fountaine, 2009). This inconsistency has in many cases hindered their establishment as proper components of disease control programs in crop production.

In summary, it is highly encouraging that a good disease protection was obtained in different crop species and toward different pathogens by PSP1-treatment and that in at least one case the protection was observed under natural growth conditions. These results show that PSP1 could be a valuable and sustainable alternative to chemical pesticides for successful control of a large number of diseases in important crop species, although additional field trials are required before a definitive answer to this postulation can be given.

NC, SD, EM, AC, and BW conceived and designed the experiments. NC performed the experiments in strawberry and soybean. SD carried out the optimization of the fermentation process and its scaling-up. NC and SD analyzed and interpreted the data. FB and MC carried out the analytical measurements of protein. PDP optimized the phytopathological test in soybean. RB performed the phytopathological assays in sugarcane. SS carried out the wheat field trials. NC and BW drafted the work and wrote the manuscript. AC revised critically the article. NC edited the manuscript. All authors approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.