Fusarium Species Responsible for Tomato Diseases and Mycotoxin Contamination and Biocontrol Opportunities

Abstract

For many years, Fusarium species have been known as one of the most common pathogens, causing disease and producing mycotoxins in many host species both on-field and postharvest. Tomato is among the most relevant hosts due to its economic and nutritional relevance, its plasticity to be cultivated under diverse soils and climates, and consumed fresh or processed. The most common pathogenic Fusarium species are Fusarium oxysporum f. spp. lycopersici, Fusarium oxysporum f. sp. radicis-lycopersici, and Fusarium solani. However, the species presence and prevalence depend on the globe region, and other species can be found such as F. semitectum, F. oxysporum, F. equiseti, F. falciforme, or F. striatum. Most of these species’ strains are also mycotoxigenic and can potentially contaminate tomatoes and tomato-based products with several mycotoxins. Some cases of mycotoxin contamination on tomatoes were reported and caused by different fungal species. Emerging Fusarium mycotoxins have recently been reported and gained high interest due to their increasing frequency. These mycotoxins, still not deeply studied, may constitute high-risk factors for human and animal health. This chapter is dedicated to the most relevant Fusarium spp. affecting tomato crops and the consequences of consuming mycotoxin-contaminated fresh tomatoes or industrially processed and describes some promising biocontrol measures.

1. Introduction

Tomato (Lycopersicon esculentum L.) is one of the most popular vegetal crops in the world. Originally from the Andean region of Colombia, Peru, and Bolivia, it was domesticated in Mexico and introduced in Europe approximately 500 years ago [1]. Despite in South America, the fruit was considered edible, and Mexicans used it for different culinary applications; in Europe, due to its content in alkaloids, tomato plant was first used as ornamental and medicinal [1, 2]. Only 350 years ago, it was introduced in Spanish cuisine and Italian cuisine, followed by a widespread consumption [1]. Nowadays, tomato is an essential crop in nearly half of the existing countries (114 countries) and contributing significantly to the national food supply of 155 countries, accounting for 16% of the total production of vegetable species in 2020, followed by onions (9%), cucumbers (8%), cabbages (6%) and eggplants (5%) [3].

According to FAO, the total tomato production in the world was 184.8 and 189.1 million metric tons (mT), in 2020 and 2021, respectively [4]. In these years, China was the top producer accounting for 35–36% of the worldwide harvest [3], while in Europe, Italy had the highest production (6,644,790 mT), followed by Spain (4,754,380 mT), Ukraine (2,444,880 mT), and Portugal (1,741,320 mT). In terms of yield per hectare, Portugal was the one with the highest yield (97.9 mT/ha) compared to the other three European countries, while the highest farm yield was achieved in the Netherlands (475.7 mT/ha), representing eight times the yield of China (59.2 mT/ha) [5].

However, despite decades of conventional breeding and selection, the tomato crop is still susceptible to over 200 diseases and disorders during its growing season in outdoor production or greenhouses [6, 7, 8]. Some of them are common to all the production regions, while others are specific to some countries or locations. These causal agents may be diverse, including bacteria, viruses, viroids, nematodes, and fungi [9], the last most common cause of destructive diseases and mycotoxin contamination [10, 11]. Soilborne and foliar fungal diseases are the major limiting factors for tomato production [11]. Furthermore, tomato fruits are highly susceptible to fungal contamination during transportation, processing, and storage (postharvest diseases). The fungi may enter the fruits through damaged skin, or other tissues, taking the fruit at the market to wilt and squash easily [12].

Fungi have diverse but important roles in different ecosystems, with some species being crucial for soil fertility or support for plant species development [13], growth, fitness, and tolerance to stress, pests, pathogens, or herbivory (endophytic fungi) [14], or even a food-source for animals and humans [15]. However, some fungal species can also cause a panoply of diseases in plants, animals, and humans [16]. In fact, 70% of around 20% of the crop losses are caused by fungal diseases, resulting in high economic costs [17, 18]. Furthermore, most of the fungal species can produce different primary and secondary metabolites that can have beneficial or harmful effects on the surrounding organisms, which may have a positive agricultural or medicinal value, or cause severe health issues in animals and humans, like mycotoxins do [19, 20]. The most critical mycotoxins to animal and human health are aflatoxins, fumonisins (FUM), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEN), and ergot alkaloids, mainly produced by Aspergillus, Penicillium, Fusarium, Stachybotrys, and Claviceps species [21, 22].

The main fungal genera found in tomato plants and fruits are saprotrophic, being naturally present in soil and comprising species with an important role in soil fertility, decomposing organic matter, and facilitating carbon and nitrogen flow such as Alternaria, Fusarium, Penicillium, and Aspergillus, that can be easily found mainly in postharvested tomatoes [10, 23]. However, some of these fungi can also be highly pathogenic and capable to producing mycotoxins, constituting a high risk of yield and quality losses [16]. In fact, the Alternaria genus is responsible for a decrease of 50–80% of the crop yield, causing “Early Blight” (mainly Alternaria solani) and includes mycotoxigenic species that produce tenuazonic acid (TeA) and alterotoxins [10]. Also, Fusarium spp. can cause several diseases on tomato plants and is responsible for “tomato fruit rot” post-harvesting. Furthermore, Fusarium spp. have the ability to synthesize most of the major mycotoxins, including moniliformin (MON), FUM, DON and ZEN [12, 16, 24, 25], which could potentially leading to contamination of the fruits. In tomato crops, one can find other fungal genera responsible for diseases, such as the primary saprophytic Septoria lycopersici, causing “Septoria Leaf Spot”, and Botrytis cinerea, causing “Gray Mold” [26, 27, 28, 29], the obligate parasite Leveillula taurica (responsible for “Powdery Mildew”), the phytopatogenic fungi Colletotrichum spp. (Anthracnose) and Verticillium spp. (verticillium Wilt), and the pathogenic oomycete Phytophthora infestans [8, 9, 10, 11]. Typical phytopathogenic fungi also infect tomato plants causing “Anthracnose” (Colletotrichum spp.), “Verticillium Wilt” (Verticillium spp.), “Powdery Mildew” (Leveillula taurica - an obligate fungal parasite), or “Late Blight” (Phytophthora infestans – a pathogenic oomycete), for example [8, 11]. The tomato industry relies on fresh and frozen tomatoes as raw materials being “Gray Mold” and “Soft Rot” the most important postharvest disease of tomato worldwide [7, 30, 31]. However, the distribution and frequency of the species depend on the step of the tomato food chain, with ones more likely to be found on the field and others in greenhouses [32], during processing, or storage [33]. Tomatoes for the processing industry are produced under field conditions [7], while tomatoes for fresh consumption may be produced in both field and greenhouse conditions.

Fusarium is one of the most important fungal genera affecting the main crops worldwide, including several species with capability to cause diseases and mycotoxins contamination, which constitute a high risk of yield and quality losses [16]. In tomato, Fusarium oxysporum is one example, responsible for yield losses of 10 to 80%, depending on the region [7]. Specifically, F. oxysporum f. sp. lycopersici (Fol), which is the forma specialis affecting tomato plants [34], is responsible for about 14% of the economic losses in tomato crops, being the fifth most important plant pathogenic fungus worldwide [35]. Beyond its pathogenicity, F. oxysporum is a great producer of MON [25], also producing beauvericin (BEA), enniatins (ENNs), FUM, fusaric acid, and fusarins [36]. Other species and formae speciales of Fusarium genus also cause diseases on tomato, such as F. equiseti and F. oxysporum f. sp. radicis-lycopersici (Forl), that cause “Fusarium crown and root rot” [37, 38], F. falciforme and F. solani f. sp. eumartii, responsible for causing “Fusarium foot rot,” and F. striatum, which causes “Fusarium crown and stem rot” [6]. From tomato fruits, the main Fusarium species isolated are F. oxysporum, F. solani, F. equiseti [12, 39], F. semitectum, F. subglutinans [39], F. proliferatum, and F. verticillioides [12], causing “Fusarium fruit rot” and potentially contaminating them with a panoply of mycotoxins [12, 36].

Nowadays, tomatoes are eaten fresh or processed into a great variety of products, such as sauces, ketchup, pulp, aseptic paste, juices, and canned and dried tomatoes [7, 23, 30], which may also be used as ingredients in a panoply of other foods [30]. This means that most of the population is often in contact with tomatoes and with the mycotoxins eventually present in them [33].

Despite mycotoxins contamination in fresh tomatoes is not usual, it is a real concern after processing, mostly due to the presence of Alternaria spp. that typically occurs under specific storage or processing environmental conditions that promote fungal growth, being temperature and humidity the major environmental factors [23, 40, 41]. The large consumption of tomatoes associated to the possible presence of mycotoxins may have a significant impact on human health [33]. In Europe, concerns were raised about the dietary exposure to Alternaria toxins that are chemically stable during food processing [41, 42]. In fact, there are reports of most of the tomato products contaminated with at least one Alternaria mycotoxin, whereas no Alternaria toxins were found in fresh tomatoes [43]. Recently, also Fusarium mycotoxins have been detected in tomatoes and derived tomato products, such as Fumonisin B2, one of those with higher values [23]. Other emergent Fusarium mycotoxins are ENNs A, A1, B and B1, and BEA, which are not regulated by governments worldwide [44, 45]. These emerging mycotoxins were detected in 58.8% of the tomato product samples (tomato powders, canned tomato, tomato sauce, ketchup, tomato juices, and dried tomatoes), but, curiously, not in fresh tomatoes [44, 45]. Thus, currently, there is a growing concern about the presence of mycotoxigenic fungi in tomato plants and fruits, being the control of fungi and their mycotoxins a crucial step to prevent yield losses and human health issues.

For that reason, in the last decades, some different ways of fungal and mycotoxins control have been developed, including some based on the use of biological control agents, such as antagonistic fungi, Bacillus and Streptomyces spp., endophytic bacteria and fungi, plant extracts, among others [46, 47, 48, 49, 50, 51, 52]. In fact, curiously, the concern with pests and diseases started long time ago in thethirteenth century, with Mexicans using agricultural practices as rotation and co-cultivation with different species to avoid the problems associated with high levels of moisture and certain pests [2]. For instance, red tomato was cultivated in the dry season due to its sensitivity to moisture, and husk tomato was co-cultivated with chili to decrease the incidence of pests [2].

Currently, one wide biocontrol measure against pathogens is the use of endophytic fungi, which reside in symbiosis within the plant and that is antagonist to the pathogen, disrupting its life cycle, preventing the colonization and infection, reducing the pathogen sporulation, and affecting its ability to survive [14]. However, also the endophytic fungi may be able to produce mycotoxins and to introduce them to the agri-food chain, posing a risk to the consumers [53]. For example, Fusarium is one of the endophytic fungal genera with significant potential to be used as biological control agent, comprising five or more known antagonistic species [54], such as F. solani strain K, which is known by increasing tomato defense against red spider mite infestations [55]. However, as previously mentioned, Fusarium is also a fungal genus that comprises relevant mycotoxigenic species. Therefore, to minimize the risk of mycotoxin contamination in tomatoes, following proper agricultural and postharvest practices is important.

This chapter aims to present the most relevant Fusarium species affecting tomato crops and their associated mycotoxins, as well as the consequences of consuming fresh tomato or industrially processed contaminated with mycotoxins and some promising biocontrol measures.

2. Fusarium diseases of tomato

The Fusarium genus is a very diverse group of filamentous fungi naturally present in soil and plants. It encompasses numerous species, with diverse roles in the ecosystems, since saprophytic or endophytic to phytopathogenic or mycotoxigenic ones. Moreover, some species can also cause infections in animals and humans, especially in the immunocompromised individuals [16], and most of them produce interesting metabolites with diverse applications. Its singularity and diversity turn Fusarium into one of the most relevant fungal genera in agriculture. Thus, this section is dedicated to the different tomato crop diseases caused by Fusarium species, their symptoms, distribution, and impact on production and economic yield.

2.1 “Fusarium wilt”

“Fusarium wilt” (FW) of tomato is one of the most frequent and destructive vascular disease responsible for extensive losses in the tomato crop production, both in the field and inside greenhouses, worldwide. This disease typically reduces the crop yield to minimal or absent [56]. However, yield losses are variable, with reports of 45% due to Fol in India and Cameroon [56, 57], or of 60–70% in Mexico [58].

The process of infection starts with the recognition of the roots by the fungi, attachment to the root surface, and penetration and colonization of the root cortex [34, 35]. The hyphae proliferate within the xylem vessels affecting the vascular tissue of the plant, clogging them with mycelium, spores, and/or polysaccharides produced by the fungus, which leads to the wilting symptoms, close to those of draught [34, 35, 59]. Furthermore, the fungus secretes several toxins in the vessels, such as fusaric acid, dehydrofusaric acid, lycomarasmin, and others, which, when carried to the leaves, cause reduced chlorophyll synthesis leading to reduced photosynthesis and decontrolled water loss through transpiration, also resulting in leaf wilting, interveinal necrosis, browning, and eventually death of the plant [59]. Therefore, the disease typically starts with wilting and yellowing of the lower leaves. The leaves or the infected parts of the plant loose turgidity and become gradually light green, yellow, brown, and finally die [35, 59]. As the fungus progresses through the plant, the wilt also spreads to the entire plant, and all leaves turn brown and become necrotic up to the whole plant death [59].

FW in tomato is mainly caused by species and formae speciales of the F. oxysporum complex. This species complex is worldwide spread and includes both nonpathogenic species, as well as species with the ability to cause disease in most of the crops, animals, and/or humans [16, 35, 60]. There are around 106 phytopathogenic formae speciales of F. oxysporum well documented affecting 45 plant families, being Fol the main responsible for FW in tomato [61]. F. solani can also be found in tomato plants causing FW, but mostly in varieties with resistance to F. oxysporum [62].

Fol was first described by Massee in 1985 in England [63] and was already documented in more than 30 countries [12, 58, 64], in several different environments and climates, from the dry and hot tropics up to the temperate climates [65], being the high temperatures and sandy and/or acidic soils the preferred conditions [66]. Currently, according to the tomato plant resistance genes that it overcomes, Fol comprises already three pathogenic races (race 1, race 2, race 3) [61], being expected a new race (race 4) until the end of 2023, considering the time observed between the appearance of two consecutive races [64]. Race 1 was described in 1886 and race 2 in 1945, in Ohio, having a worldwide distribution nowadays, while race 3 was firstly observed in Australia in 1978 and is present in a more limited region [35, 65]. Today, most of the commercial tomato varieties are resistant to races 1 and 2, but only few are resistant to race 3 [35], which results in higher yield losses.

Across the United States, there are reports of all three known physiological races of Fol, with the race 3 causing up to 80% of the disease incidence in tomato varieties resistant to races 1 and 2, whereas, in China, Fol race 1 is the mostly present but race 2, only reported in Zhejiang causes yield losses between 30 to 60% [7]. In Italy, FW is also mostly caused by Fol race 1 and race 2 and considered a major cause of economic loss in tomato production [7]. Also in the Southern Hemisphere, FW caused by Fol is a concern, being widely present in Brazil and Australia, and an emerging pathogen in Chile, causing huge yield and economic losses [7].

2.2 “Fusarium crown and root rot”

“Fusarium crown and root rot” (FCRR) is also a major fungal disease of both field and greenhouse tomato production [7].

FCRR is characterized by early loss of cotyledons, newest leaves, and basal stem necrosis [37]. Symptoms include wilting, where the leaves and stems become limp and lose their turgidity, yellowing of the leaves, due to a reduced ability to take up nutrients and water, stunted growth, with reduced overall plant size and vigor, because of the roots start to be darkening, browning, and rotting, losing the ability to support the plant’s growth, while the crown shows lesions as dark and sunken areas on the stem at the soil line [37, 67]. The disease incidence and symptom severity increase with the inoculum concentration resulting, in the most severe cases, in plant collapse and death [67].

This disease is caused by both Forl, first reported in Japan in 1974 [7, 37], and by F. equiseti [38]. Forl is the main responsible for causing FCRR in tomato, but, contrary to Fol, is reported as a pathogen also in other plant hosts, such as eggplants, Fabaceae spp., and Cucurbitaceae spp. [61], and has no races identified [37, 61]. However, there are ten vegetative compatibility group (0090–0099) identified for Forl isolates from Israel, Belgium, Canada, Greece, France, Italy, Japan, and the United States, which can be divided in different subgroups [37]. F. equiseti is primarily a saprophyte or secondary a root colonizer, cosmopolitan in areas ranging from cool and temperate to hot and arid, which can affect a large range of plant hosts and even be an allergen or pathogen to humans [68]. Contrary to F. oxysporum, F. equiseti has not formae speciales reported.

FCRR causes losses estimated at up to 90% and 95% in Tunisian and Canadian greenhouses, respectively [69] and is present in all the USA tomato fields [37], causing a tomato yield reduction of 15–65% in Florida [70]. Also, in Italy, this disease causes substantial yield loss in both greenhouse and soil-less production, with a up to 60% mortality rate during severe Forl outbreaks [7] and being present in 24% of the greenhouses of Sicily and 66% of greenhouses in Sardinia during the 1990s [7]. Forl is also becoming an increasing threat in Brazil, with disease indices ranging from 10 to 50%, and Chile, affecting fresh tomato production more severely [7].

In Australia, Forl was not reported yet, but F. oxysporum strains were collected producing symptoms of rot on tomato roots and crowns such as those caused by Forl, but with different growth temperature, a larger host range, and variable pathogenicity, being this disease named as “chocolate streak disease” (CSD), different from FCRR caused by Forl [7].

2.3 “Fusarium foot rot”

“Fusarium foot rot” is a disease that is more prevalent in tomato crops grown during the cooler months and that usually occurs in patches in low-lying areas of the field [62]. Despite this disease does not often kill the tomato plants, it can substantially reduce the yield, up to 80% [62].

The fungus enters the cortical tissues of roots and hypocotyl by the existing wounds and causes brown lesions that extend into the vascular system, up to 25 cm of the root-stem transition zone. Despite the fungus kill the young seedlings, the infected plant usually does not wilt until its first full fruiting condition, and after fruit harvesting, or if atmospheric and other conditions reduce transpiration, the symptoms can reduce [71]. The symptoms may include varying degrees of chlorosis, mottling, and necrotic spotting on young foliage, and in severely affected plants, the main root and the crown can also be rotted [62, 72].

“Fusarium foot rot” in tomato can be caused by Forl [71], or by species and formae speciales from F. solani complex (FSSC), namely F. falciforme [72] and F. solani f. sp. eumartii [62]. F. falciforme was erstwhile called Acremonium falciforme, but, based on the results of more recent morphological and molecular phylogenetic analyses, it belongs to the F. solani complex now [73]. This species complex is easily isolated from soils in a variety of environments, comprising pathogenic species of a large number of plant species, including other economically important crops and trees [68]. F. solani f. sp. eumartii was firstly reported by Carpenter in 1915 in Pennsylvania, causing “Eumartii wilt” in potato [74]. However, this forma speciales of F. solani can also cause foot rot, tuber dry rot tubers and postharvest diseases of potato, foot rot of tomato, and stem and collar rot of eggplant and sweet pepper [74]. In the same way, F. falciforme can cause root rot in melon [75], lima bean [76], onion basal rot [77], root and stem rot in papaya [78], fruit rot of watermelon [79], and foot rot in tomato [72]. Furthermore, the FSSC comprises some of the most important Fusarium agents of animal and/or human infections in immunocompromised individuals [16, 68, 80]. For example, F. falciforme caused vertebral abscess and osteomyelitis in a 53-year-old woman with an autoimmune disorder [81].

2.4 “Fusarium crown and stem rot”

“Fusarium crown and stem rot” is a recent disease of the tomato crop, although it appeared in 2006 in commercial greenhouses of tomato plants in Canada and USA. Initially, pathologists were not able to identify the causal agent species, only knowing that it belonged to the FSSC. Only in 2014, the Fusarium isolates were identified as F. striatum, based on morphology and molecular analysis [6]. Recently, in 2020, it was also reported the presence of this disease since 2015–2016 in greenhouses in two states of Mexico [82].

“Fusarium crown and stem rot” is characterized by the development of dark brown cankers on stems and brown discoloration of the pith area. Symptomatic plants have brown lesions at the graph connections and at the pruning sites that evolve as cankers and lids to the dead of the plants within few weeks (8 weeks) after the appearance of the symptoms. The cankers are flat and depressed with a clear delineation from the stem. Plants with stem rot show chlorotic and senescent wilted leaves, and the whole plant exhibits wilting [6, 82]. In the base of diseased plants, the development of orange-red to dark-red perithecia on necrotic tissues and on rockwool slabs usually occurs. Plants can survive longer if the canker is removed within 2 weeks after the first symptoms were observed. Moreover, this new disease caused reminiscent of infection by Forl. A very typical symptom that allows to distinguish this disease from that caused by Forl is the discoloration of the pith rather than the vascular tissues that remain untouched [6]. Another distinguishing feature, but for the disease caused by F. solani f. sp. Eumartii, appears to be the homothallism of F. striatum in contrast to the heterothallism of F. solani f. sp. Eumartii [6, 83].

Several species for the FSSC are saprophytic, but there are also some that are phytopathogenic, causing severe losses mainly on horticultural crops. Within six years, from 2006 to 2012, F. striatum infected plants increased up to 20% in North America [6]. In greenhouses in Mexico, as the fungal infection is quite recent, the incidence was only up to 5% [82]. F. striatum was first described by Sherbakoff in 1915 who isolate the fungi from potato tubers originated in Colorado. A distinct characteristic is the formation of a “Striate rot” when inoculated in potatoes [6].

F. striatum in tomato was only reported twice, but it has other hosts, such as Panax notoginseng, where the first appearance was reported in 2020 in China [84]. In ginseng, F. striatum is an endophytic fungus with the ability to cause root rot disease. The isolate obtained from embryos could cause root rot in plants, which is a very interesting case of possible interchangeable functions, either as endophyte or as a pathogenic fungus. This situation poses difficulties during cultivation, where the fungus cannot be effectively prevented because it is a seed-borne pathogen. So far, such behavior has not been reported in tomato, but further studies should be conducted to understand the function of F. striatum in tomato seeds as the reported presence of the fungus in tomato plants was always in greenhouses.

2.5 Fusarium tomato fruit rot

“Tomato fruit rots” are important postharvesting diseases that compromise the quality of tomatoes and their possibility of being commercialized or consumed, resulting in economic losses and waste, that can reach over than 50% of the product during severe infections: 25% at harvest and 34% during transportation, storage, and market shops [85]. There are two types of tomato fruit rots: soft rots, such as “Fusarium tomato fruit rot” and dry rots. Soft rots of tomato fruit can be caused by Erwinia carotovora, Rhizopus oryzae, R. stolonifera, and Fusarium spp. and contribute to 85% of the overall loss of tomato [85].

“Fusarium tomato fruit rot” is characterized by soft rots that can extend into the center of the fruit. Usually, while the infected tissue is discolored and pale brown, the rotted tissue becomes covered by white, yellow, or pinkish mycelium [39]. Improperly handling, packaging, storage, and transportation of tomatoes promote the penetration of the fungus into the epicarp and their deposit in the fleshy parts of the mesocarp. Then, the fungus uses the ascorbic acid and soluble sugar contents in the fruit and proliferate, resulting in an enlarging necrosis of the epicarp toward the outside and in a more and more water-soaked fruit. The fungus continues extending deep into the seed cavity, destroys the seed coat and cotyledons, and eventually, the fruit completely rots [39].

The main species isolated from tomatoes with “Fusarium tomato fruit rot” are F. solani [12, 39], F. oxysporum, F. equiseti [12, 39, 85], F. semitectum, F. subglutinans [39], F. proliferatum, and F. verticillioides [12]. As previously mentioned, F. oxysporum, F. solani, and F. equiseti are also associated with other tomato diseases, but usually it is not the case of F. semitectum, F. subglutinans [39], F. proliferatum, and F. verticillioides. These species are mainly found affecting other crops, for example, maize or sorghum, being not considered common tomato pathogens, affecting mainly the postharvested tomato fruits. Their distribution is wide, and they can be found in different agricultural and nonagricultural substrates. For example, F. semisectum is commonly found in both soil and plants, not confined to just subtropical and tropical regions but also present in the Arctic and arid deserts. Furthermore, it is often associated with the development of storage rot in various types of fruits, including bananas and mushrooms. Additionally, all these four species can be allergenic to humans and cause severe infections with reduced treatment options due their resistance to most of the clinical antifungals [68]. Fusarium solani, F. semitectum, and F. oxysporum, including Fol, are the most abundant species isolated from tomatoes with “Fusarium fruit rot”, while F. verticillioides, F. proliferatum, F. subglutinans, and F. equiseti represent a minor [12, 39].

3. Fusarium mycotoxins on tomato, their relevance, and control

3.1 Fusarium mycotoxins on tomato

Fresh tomatoes are rarely contaminated with mycotoxins, but their storage, even at low temperature, and processing may promote fungal growth and contamination with mycotoxins [43, 86]. As tomatoes have high water and nutrient contents, they can easily be contaminated with fungal species from Aspergillus, Alternaria, Fusarium, and Penicillium genera [45]. Thus, aflatoxins and ochratoxins produced mainly by Aspergillus spp., and FUM and fusaric acid, mainly produced by Fusarium spp., are mycotoxins that can potentially appear in tomatoes and their products. For example, aflatoxins were detected in rotten tomatoes commercialized in Nigeria [87]. Beyond these, some other emerging mycotoxins have been reported in tomato products, including alternariol monomethyl ether (AME), alternariol (AOH), and TeA, produced by Alternaria spp. [43], and BEA, ENNs, and MON produced by a wide variety of Fusarium spp. For example, TeA appears to be omnipresent in tomato products [43], and diverse assays report one or more of these emerging mycotoxins present in most of the tomato products analyzed [43, 45]. Therefore, frequent contamination of food and feed, including in tomatoes and tomato products, with emerging Fusarium and Alternaria mycotoxins has increased the interest about them [43]. These mycotoxins can provoke a wide range of toxicological effects to protect the plants, acting as bactericide, fungicide, antihelmintic, and insecticide, but they also may provoke phytotoxicity and cytotoxicity through mitochondrial modifications [88], decrease of the functionality of photosystems I and II leading to the reduction of photosynthetic pigments in the tomato leaves and to leaf wilting, cell dead, and necrosis [59].

As previously mentioned, the Fusarium species more frequently find in tomato plants are F. oxysporum, F. equiseti, F. solani, F. falciforme, and F. striatum, while in tomato fruits are F. oxysporum, F. solani, F. equiseti, F. semitectum, F. subglutinans, F. proliferatum, and F. verticillioides. However, if fresh tomatoes rarely show mycotoxin contamination because it occurs mostly during storage and processing, the main responsible species by mycotoxin contamination should be the ones found in tomato fruits. Table 1 summarizes the mycotoxins produced by these species, and it is possible to see that all the most common species found in tomatoes produce BEA and/or MON, which justifies the emergence of these mycotoxins. Moreover, F. oxysporum, which also infects the tomato plants, and F. proliferatum are producers of ENNs, being a reason for its emergence in tomatoes and tomato products.

Fusarium species	Mycotoxins produced	References
F. oxysporum	BEA, ENNs, MON, FUM, fusaric acid, fusarins, bikaverin, and isoverrucarol	[25, 36, 68]
F. solani	MON, fusaric acid, and fusalanipyrone	[36, 68]
F. equiseti	BEA, MON, butenolide, equisetin, fusarochromanone, ZEN, trichothecenes, diacetoxyscirpenol, nivalenol, and T-2 toxin	[36, 39, 68, 89]
F. semitectum	BEA, MON, trichothecenes, ZEN, apicidins, equisetin, fusapyrone, sambutoxin, and diacetoxyscirpenol	[36, 39, 68, 89]
F. subglutinans	BEA, MON, fusaproliferin, fusaric acid, and FUM	[36, 39, 68]
F. proliferatum	BEA, ENNs, MON, fusaproliferin, fusaric acid, fusarins, and FUM	[36, 68]
F. verticillioides	BEA, FUM, fusaric acid, and fusarins	[36]

Table 1.

Summary of mycotoxins produced by the Fusarium species reported in tomato. Fruits.

3.2 Fusarium mycotoxins relevance in human health

The large consumption of tomato products associated to the emergence of mycotoxins in them may have a significant impact on human health [33], resulting in acute or chronic consequences such as carcinogenic, teratogenic, immunosuppressive, or estrogenic issues [21]. In this way, the most common and known as harmful mycotoxins are regulated by the European Commission that set the maximum levels allowed of each individual mycotoxin [90]. These include aflatoxins, FUM, OTA, DON, ZEN, and ergot alkaloids [21, 22]. These limits turn all the products containing high levels of mycotoxins strongly devalued or even banned from being sold. However, the in vivo toxicity and toxicokinetic data about the three emergent mycotoxins in tomato (MON, BEA, and ENNs) are limited, as well as the data of their occurrence in food [43], which means that these mycotoxins are not regulated by governments worldwide, yet [45].

In vitro studies suggest that ENNs A, A1, and B1, BEA, and MON have genotoxic and immunomodulating effects, BEA and ENN B can cause a reproductive health hazard, and MON can cause severe toxicity in animals, mainly affecting the heart [43]. This might be a health concern, especially among children and the populations consuming a high amount of these products [45].

On the other hand, FUM, DON, and ZEN, also mainly produced by Fusarium species, are known as harmful to human health [16]. For example, FUM B1 is genotoxic and a cancer promoter, while ZEN is an estrogenic mycotoxin, being linked with endometrial adenocarcinoma and hyperplasia [21, 91]. Also, in an essay with rats, DON-induced DNA damage and the expression of proteins related to inflammatory response implied the apoptotic pathway [92].

As previously mentioned, one or more mycotoxins have been found in most of the tomato products analyzed in diverse essays. This multi-mycotoxin co-occurrence is, in fact, a frequent issue that may result in synergistic, additive, or antagonistic effects of mycotoxins. For example, when aflatoxins are present together with DON and T2 toxin, OTA with FUM, or FUM with DON, there is a synergetic effect of these mycotoxins [21, 93, 94]. Thus, it is urgent to consider the toxicological effects of mycotoxins mixtures in the new risk assessments [94].

Considering, the emergence of mycotoxins in tomato products and the lack of knowledge about their real effects on human health; currently, there is a growing concern about the presence of mycotoxigenic fungi in tomato plants, fruits, and products, being the control of these fungi and their mycotoxins a crucial step to prevent both yield losses and human health issues.

3.3 Mycotoxins control measures

All stages of production, harvesting, transport, storage, and processing of tomatoes might undergo to mycotoxin contamination [45]. However, since mycotoxins are not usually found in fresh tomatoes, the storage and processing of tomatoes appear to be the most critical stages.

Nonetheless, within the food supply chain, similar to the feed supply chain as both are integrated, mycotoxin management must be comprehensive, encompassing all phases from the cultivation of raw materials until the production of food [95]. During tomatoes production, good farming practices should be applied to minimize the mycotoxigenic fungi presence. Postharvesting during transportation, storage, processing, sanitation and controlling humidity, temperature, ventilation, insects, and rodents presence are also good practices to avoid fungal proliferation and mycotoxins production [95, 96]. It is also important to keep in mind that most of the mycotoxins are chemically stable to heat during processing [45].

There are also new strategies that may be used, inclusive in combination, such as using bio-competitive fungi as biocontrol agents, varieties resistant to pathogens, apply antibody-mediated technology, or even nanomaterials engaged for antifungal or inhibition of mycotoxins [95]. Also, some physical, thermal, and biological methods may be used such as cleaning and aggressive sorting, nonionizing and ionizing irradiation, or microorganisms with detoxification activities [95, 96]. Other methods to control fungal and mycotoxin contamination have been studied, such as ozone application, that inhibits fungal growth and cold plasma technology, which reduces and degrades mycotoxins in food and feed, pulsed light, radio frequency, and microwave for reducing mycotoxin contamination [95]. Additionally, some binding agents (inorganic or organic) or bio-transforming agents (e.g., bacteria, yeast, fungi, and enzymes that degrade mycotoxin molecules in nontoxic metabolites) may be used to control fungal proliferation and mycotoxin contamination [95, 96].

Nevertheless, despite the availability of various strategies to manage mycotoxins by either pre-harvest prevent or post-harvest eliminate/decontaminate, achieving their total eradication remains very difficult. The precision of detection methods plays a fundamental role on the control of contaminations [45, 95].

4. Management of diseases caused by Fusarium spp.

Some good farming practices can help reducing the risk of disease development and minimize the severity of fungal diseases. However, it is important to note that managing Fusarium diseases can be challenging as the pathogen can persist as chlamydospores in the soil or as mycelium in infected plant debris for long periods [97]. Thus, a combination of practices, including prevention, sanitation, single-gene resistance, and cultural management is usually the most effective approach to control the disease and minimizing the impact on tomato crop.

Prevention may be accomplished firstly by choosing an adequate planting site. This should be characterized by a well-drained, not sandy or acidic soil, without high temperatures or high fluctuations between day and night, and with good air circulation [95, 96]. For instance, Fol is favored by high temperatures in the soil, but F. solani may be favored by a temperature of 18°C or 28°C depending on high or low soil moisture, respectively [98].

Secondly, it is recommended to utilize effective irrigation systems such as drip irrigation. This approach prevents leaf sprinkling and excessive watering [30], which could potentially disperse the pathogen over greater distances [66]. Also, giving proper spacing between contiguous plants and doing a proper fertilization avoiding excessive nitrogen are wise [95, 96].

Seed sanitation by means of several different treatments as heat or thermotherapy may decrease seed-borne infections. In fact, efforts have been undertaken to manage Fusarium spp. by regulating not only the quality of propagating plant material like seed and seedlings, but also the quality of planting substrates, trays and other items used in nurseries and greenhouses [99].

Furthermore, implementing an integrated pest management strategy and choosing tomato varieties that exhibit resistance to specific Fusarium species and races represent straightforward, cost-effective, and efficient preventive measures [58]. In fact, plant resistance to pathogens is theoretically the most economical, effective and environmentally friendly approach to disease management [37, 100]. However, no variety was so far completely resistant to, for instance, Fol. Akram and co-workers (2014) [97] verified that only three varieties were moderately resistant to Fol, being all the others either susceptible or very susceptible. Other authors reported several varieties resistant either to all races of Fol and susceptible to Forl or resistant to combinations of two races of Fol and to Forl [37]. For each of the three Fol races, resistance has been introgressed from wild tomato species, using four R genes [100]. The relationship host-pathogen is under constant modification/adaptation with the pathogens gradually overcoming the tomato cultivar resistance mechanisms. Plant breeding had been limited success due to the emergence of new pathogenic races [101], combined with the lack of knowledge on the mechanisms underlying the host-pathogen interaction, which may evolve to infection or to the resistance of the host [100].

Doing a good soil management is also important, namely doing crop rotation with non-susceptible plants, which can help reducing the fungal population in the soil that is pathogenic to tomato crops [95, 96], or even opting for soil-less systems, which offer a unique opportunity to use antagonists. Biological control of Fusarium diseases by means of action of nonpathogenic strains of F. oxysporum may be easier with its application in soil-less cultures of tomatoes than on soil cultures, considering the need of high mass-production of the antagonist and its incorporation in an appropriate formulation to be applied on several hectares [47]. Investing in sanitation is also important to limit the disease’s spread and can be done, for example, removing and destroying infected plants and choosing seeds with high sanitary quality, since seeds may be vectors of the fungi at long distances [66].

Employing fungicides is a widespread practice, but it frequently fails to meet the expected levels of effectiveness due to the presence of fungi in both the soil and seeds, which poses a challenge [97]. Fol has been controlled by disinfection of the soil with chemicals as methyl bromide with damaging effects on the environment and on human and animal health [102]. The extensive use of fungicides, beside having negative impact in the environment, promotes the emergence of resistant strains and may kill the endophyte fungi that collaborate with the plant and ensure its virility [16]. Furthermore, this measure needs to be applied several times and in the proper time to be effective as a preventive method, which implies significative economic costs [101, 103]. To prevent and minimize these negative impacts, research on new and biological alternatives to chemical fungicides is a rising issue, including the use of bacteria or other fungal species [24, 54] for biocontrol of harmful species. The simultaneous use of biocontrol agents and synthetic fungicides [54], as well as genetic engineering, for example, introducing genes, which encode proteins with antifungal activity into the plant genome, are alternative approaches that can be used in some cases [100, 104].

4.1 Biocontrol measures

Biological control has received increasing attention and acceptance in the last decades. It aims to control pests and diseases importing, reinforcing, or conserving other organisms (biocontrol agents) in the environment [7]. Moreover, its high efficiency, target-specificity, and self-sustainability minimize the public concerns of impacts on health and environment, resulting in a higher public acceptance and advantages compared to the conventional methods [7].

To biocontrol fungal plant diseases, a mix of different strategies can be applied. Antagonist species can be used to produce inhibitory metabolites, act as parasites on pathogenic fungi, or compete with pathogenic species [7, 54, 105]. Some of the most used biocontrol agents are Aspergillus spp., Chaetomium spp., Glomus spp., nonpathogenic Fusarium spp., Trichoderma spp., Penicillium spp., Bacillus spp., Pseudomonas spp., Streptomyces spp., or Serratia spp. [106]. Biocontrol of Fusarium spp. associated diseases is a sustainable and environmentally friendly approach to mitigate the impact of the pathogens on the tomato crop, on the ecosystem, and on the consumers. This approach is an alternative to conventional fungicides and fumigation by methyl bromide, which are known by their negative effects on the human health, animal well-being, and environment (e.g., depletion of the ozone layer) [107].

For example, nonpathogenic Fusarium species and fluorescent Pseudomonas are reported as the more effective and consistent biocontrol agents against FW in tomato produced in natural soil, while strains from Penicillium, Streptomyces, and Aspergillus species are mostly effective in vitro conditions [7].

Also, metabolites produced by these and other organisms may induce the plant defense response against pathogens, reducing the effects of disease [7, 54, 105].

In tomato, there are reports of bacteria, fungi, and plant and algae extracts effectively used as biocontrol agents against diseases caused by Fusarium spp. [7]. In fact, some Fusarium strains affecting tomato are known as having poor competitive fitness against other microorganisms, such as Forl, being the biocontrol using antagonistic organisms effective. Thus, several antagonistic microorganisms have been successfully tested against Forl and Fol [7]. However, around 79% of the tests on tomatoes were conducted in greenhouse conditions, and only 12% in the field condition, which means that further field tests are necessary [7].

4.1.1 Bacteria as biocontrol agents against Fusarium diseases in tomato

Some soilborne bacteria, such as Pseudomonas, Bacillus, or Streptomyces spp., for example, can be used as competitors against Fusarium species.

Some of the most relevant Pseudomonas species used as biocontrol agents in tomato are P. fluorescens, P. chlororaphis, and P. putida. For example, P. chlororaphis can reduce the incidence of FCRR in 44–60%, and P. putida can reduce the incidence of FW in 41–94% [37]. P. fluorescens was also applied to control FW with success, what was concluded by the reduction of the incidence in 53–72% or 65–85%, depending on the production system, in greenhouse, or in field, respectively [37], and, in vitro, P. fluorescens can suppress the growth of Forl synthesizing the antibiotic 2,4-diacetylphloroglucinol [7]. P. fluorescens can also be used combined with the fungicide benomyl to decrease FW incidence more effectively in greenhouse environment [108].

From Bacillus genus, some of the most relevant species used as biocontrol agents in tomato are B. subtilis, B. amyloliquefaciens, B. cereus, and B. pumilus [109]. In India, B. amyloliquefaciens and B. subtilis were reported as capable to reduce the incidence of FW in 44–46% and 53–64%, respectively, under field conditions [37]. In vitro, B. amyloliquefaciens also can inhibit the growth of F. solani by 95.2% and B. cereus reduced the growth of the fungus in 55.7%. Also, B. pumilus and B. subtilis can inhibit F. solani growth in 70.46% and 82.1%, respectively [109]. The same biocontrol potential was verified in vivo, with B. cereus controlling 81.2% of the disease, B. amyloliquefaciens controlling 75%, and B. pumilus and B. subtilis controlling 62.5% individually [109]. Some strains of B. pumilus (e.g., ToIrMA) also can reduce FW incidence in about 73% and increase the root and shoot length 60% and 84%, respectively [49]. While some B. subtilis strains (e.g., QST713 and EU07) can reduce the FCRR incidence by 52% and 75%, respectively [110].

Also, B. subtillis can be used combined with a fungicide, such as hymexazol or the resistance elicitor acibenzolar-s-methyl, to produce greater reductions in the FCRR disease incidence than any of these treatments alone [111].

Endophytic bacteria also can be used as biocontrol of FW and FCRR, including species from other plants neither Solanum lycopersicum. For example, essays using endophytic bacteria from Solanum sodomaeum, Solanum bonariense, and Nicotiana glauca reported their capability to control Fol [50, 51]. Also, endophytic Streptomycesspp. strains (e.g., SNL2) isolated from roots of native plants can be effective against Forl, reducing the FCRR incidence in 75.3%, and significantly increasing the dry weight, shoot, and root length of tomato seedlings [48]. In the same way, Streptomyces griseus can reduce the severity of FW in 57% in greenhouse conditions [37]. Any plant rhizosphere may carry antagonistic bacteria that can suppress pathogens, as well as enhance tomato plant growth [112]. For example, some strains of B. subtilis (e.g., BsTA16) and Acinetobacter calcoaceticus (e.g., AcDB3) have potential against Fol race 3 or Forl in tomato, respectively [112]. Another example is Achromobacter xylosoxidans that can reduce the incidence of FW in 50% in greenhouse environments [37].

4.1.2 Fungi as biocontrol agents against Fusarium diseases in tomato

The fungal high reproductive rate, short generation time, and high capability to survive both in the environment, such as parasites or saprotrophs, and in the plant, such as endophytic, turn fungi an interesting, potential, and sustainable option to biological control [14, 54]. In one hand, these species can use competition for space and nutrition, volatile and diffusible antibiosis, and hydrolytic enzyme production to minimize the pathogen inoculum. In the other hand, they can also colonize the plant roots, to induce growth and nutrient adsorption for the plant, and invade the vascular tissue or epidermal cells of plant root, to induce systemic resistance in plant, and defend it from the pathogen infection [105]. For example, nonpathogenic strains of fungi can be used as competitors against Fusarium species causing FW, helping suppress this disease, like nonpathogenic isolates of F. solani, or certain Trichoderma species, for example [7, 68, 105].

Trichoderma is one of the greatest well-known fungal biocontrol agents against plant fungal diseases, comprising 25 biocontrol agents [54, 105]. In tomato crops, Trichoderma asperellum MSST is an efficient promising biocontrol agent against Fol, inhibiting its entrance in the plant root, inducing the systemic resistance in the plant, and reducing the FW incidence up to 85% [105]. Another Trichoderma species known for its effectiveness against Fusarium diseases is T. harzianum. When used as a seed coating or mixed with wheat-bran/peat for tomatoes cultivated in fields infested with Forl, it led to a remarkable 26.2% increase in yield [113]. Research carried out in Thailand, Israel, Egypt and the USA has demonstrated that the application of T. harzianum leads to a significant decrease in both the disease incidence and severity. The reduction in incidence ranged from 30% to 80%, while severity reduction varied between 24% and 44%. These variations are influenced by the specific region, although there is some overlap in the observed values [37]. T. harzianum can also be applied combined with Rhizophagus intraradices, with Aspergillus ochraceus and Penicillium funiculosum, or with Pseudomonas sp. and R. intraradices, reducing, even more, the severity and incidence of these diseases [37, 114].

Other Aspergillus and Penicillium species with biocontrol application are A. nidulans, A. awamori, and P. digitatum, which can reduce the severity of FW by 63%, 37%, and 21%, respectively [37].

Using endophytic fungi is also a wise measure of biocontrol since it resides on the natural symbiosis established within the plant between the antagonist and the pathogen. The antagonist disrupts the pathogen life cycle, prevents the colonization and infection, reducing its sporulation, and ability to survive, and therefore protecting the plant [14]. For example, nonpathogenic endophytic F. solani strains may reduce the incidence of diseases caused by Forl by 47%, or even more if combined with certain fungicides [115]. Also, nonpathogenic strains of F. oxysporum can induce the systemic resistance and defense reaction of the tomato plant and successfully control Fol [7] and Forl, reducing the severity and incidence of FW in 38–58% and 57–78%, depending on the production done in greenhouses or in field condition (respectively), and the incidence of FCRR in 78% in greenhouses [37].

Other fungi also used as biocontrol agents are Chaetomium lucknowense and C. globsum. Both can reduce the severity of FW in 36% and 44% [37]. Additionally, Pythium oligandrum can control Forl and FCRR in tomato [54]. Also, Rhizoctonia strains can reduce completely the vascular discoloration caused by Forl on tomatoes in greenhouse conditions and up to 70% in the field [116], and some mycorrhizae species, such as R. intraradices, can effectively reduce the severity of FW in GH (72%), as well as the incidence and severity of FCRR in field (18–71% and 16–53%, respectively) [37].

4.1.3 Other biological solutions against Fusarium diseases in tomato

Biopesticides, that is. natural products derived from living organisms, such as plants, extracts, and algae with fungicidal properties, are being studied and developed to control pests in agriculture and forestry with reduced reliance on synthetic chemicals [52, 117, 118].

Aqueous extracts from plants such as, for instance, garlic and the tropical plants Callistemon citrinus, Cymbopogon citratus, and Oxalis barrelieri, have anti-FW potential properties by inhibiting mycelial growth or by stimulating the tomato defense system [52, 117]. The disease incidence decreased in treated plants with percentages of reduction ranging from 51–84% depending on the treatment [52]. The authors concluded that these compounds will be helpful in the management of the disease caused by Fol even under field conditions [117] improving productivity and the quality of the production [52].

Macroalgae are also very promising organisms against wilt disease in tomato as they contain several bioactive antifungal complexes that can be used in the field [118].

Essential oils extracted from Cymbopogon citratus and Ocimum gratissimum were very active against radial growth and conidia germination of F. oxysporum with 100% inhibition of mycelial growth. The inhibitory effect is due to the presence of phenols, flavonoids, tannins, and coumarins [56]. Neem oil, which is derived from the neem tree, was the essential oil that had the strongest inhibition on F. oxysporum comparatively to garlic oil and argan oil due to the presence of azadirachtin, a terpenoid with antifungal activity, reaching 40.54% [119]. Linalool is also a common volatile monoterpene alcohol found in plants with a wide range of fungicide activity, namely against the soilborne Forl. The remaining doubts that still hamper its wide use in the field are the effects on soil-beneficial microorganisms [120].

All these findings are contributing for the development of “green” fungicides.

Organic amendments are another group of biocontrol agents that can be used against Fusarium infections in tomato. For example, the combination of pelletized poultry manure with heating or solarization can significantly reduce the FW severity [121]. Also, the combined application of biocontrol organisms and amendments can result in higher biocontrol efficiency of diverse genera of bacteria or fungi, excepting Pseudomonas and Penicillium [7].

Herbal vermicompost and vermicompost biofortified with selected biological control agents (such as T. harzianum, P. fluorescens, and B. subtilis) significantly increased soil pH, ammonium nitrogen, soil organic matter, and dissolved organic carbon, providing abundant antagonists and promoting beneficial bacteria that enhanced plant growth, yield, and nutritional quality, which, in turn, suppressed Fol effects [122]. Wang et al. (2021) [107] observed the effect of vermicompost on promoting growth of Actinobacteria, Chloroflexi, Saccharibacteria, and Planctomycetes and inhibiting growth of Proteobacteria, Gemmatimonadetes, Firmicutes, Verrucomicrobia, and Cyanobacteria. This alteration in the relative presence of Ascomycota and Basidiomycota reduced the incidence of Fol by 36.5% up to 73.9%, being the protective effect increasing in proportion to the rate of application [123]. In this way, vermicompost has still to be tested and exploited for large-scale field applications.

5. Conclusions

Within the tomato crop, the prevalent pathogenic Fusarium species include Fol, Forl, and F. solani. These particular species are responsible for inducing a range of diseases such as vascular wilts, root, stem and crown rots, and cankers, which have a global impact on both field-grown and greenhouse-cultivated tomatoes. Fusarium fungi survive as chlamydospores for long periods in the soil and wilt strains colonize the root cortex of some non-host plants, which behave as natural reservoirs of propagating structures. Thus, controlling Fusarium is challenging. Currently, single-gene editing and preferably dual-gene CRISPR/cas9 already demonstrated the ability to improve tolerance/resistance to fusarium wilt, but experimental field evaluation is still needed.

Fusarium species produce a range of mycotoxins, such as FUM, DON, and ZEN, which harm animal and human health. The wilt species, beside clogging the vessels, also can produce mycotoxins, such as fusaric acid, lycomarasmin, and dehydrofusaric acid, being fusaric acid a contaminant in tomatoes and the tomato-based products. Furthermore, most of the Fusarium spp. found in tomatoes can produce BEA, MON, and/or ENNs, which have been detected more frequently and of which effects in human health are unknown. Therefore, these fungal species have a great impact on the international economy, food safety, and security.

Further studies are needed to develop tools and models to early detect the diseases, to control the fungi, and to mitigate their effects. Under the current climatic change, a new challenge is related with the emergence of exotic new species and their associated mycotoxins in tomato and tomato-based products.

Acknowledgments

FCT - Fundação para a Ciência e Tecnologia, I.P. supported D.S. by the Ph.D. fellowship UI/BD/154444/2022. This work was also supported by FCT, Portuguese Foundation for Science and Technology through the R&D Unit, UIDB/04551/2020 (GREEN-IT, Bioresources for Sustainability).

Conflict of interest

The authors declare no conflict of interest.