Plant extract as a strategy for the management of seed pathogens: a critical review

Seeds associated to fungal pathogens are efficient vehicles for disease dissemination in the field. Such pathogens affect the seed quality and longevity, causing a decrease or loss of germination, discoloration, necrosis, and decay, in addition to leading to the production of mycotoxins in some pathosystems. To control them several synthetic chemicals are used. Nevertheless, the use of synthetic chemicals poses a risk to human health and the environment. Therefore, there is a growing demand for the use of alternative methods for the treatment of seeds, such as plant extracts. This review evaluated the use and efficacy of plant extracts for the control of fungal pathogens associated to seeds. Some control methods are used in seed treatment, plant extracts stand out due to the secondary metabolic in their constitution, which inhibit pathogen growth. The literature review showed that 100% of the studies reported that plant extracts were efficient to control the different pathogens evaluated, 63% stated an increase in seed germination, 21% reported no change in germination, 5% mentioned negative interference, and 11% did not evaluate the use of plant extracts. The aqueous extracts were used as extractors in 72% of the studies. Plant extracts were reported as promising to replace synthetic fungicides in 33% of the studies; however, 67% did not compare their use. Nevertheless, efficient extraction methods are required, considering low persistence and volatilization of plant extracts in the field. Plant extracts are efficient to control fungal pathogens.


Fungal pathogen damage in seeds
Seeds can be pathogen vectors that contaminate disease-free areas (Baker & Smith, 1966;Bisen et al., 2014;Shade et al., 2017). Pathogens can occur on the seed surface or inside the tissues, characterized as endophytes (Barret et al., 2015). In leafy vegetables, contamination of a small number of seeds is enough to trigger high incidence of the disease in the production area and Fusarium oxysporum Schltdl and Verticillium dahliae Kleb are some of the most common pathogens .
Pathogens in seeds, acting externally and internally, can cause seed discoloration, shrinkage (Gaur et al., 2020), seed abortion, rot, necrosis, decrease or loss of germination capacity, damage to seedlings as well as diseases in the later stages of plant growth (Naqvi & Rehman, 2013). When they infect the seeds internally, fungi destroy the endosperm and embryo, compromising germination and development of seedlings (Michelle et al., 2010).
According to Hendrix and Campbell (1973), fungi of the Pythium Nees genus infect seeds and seedlings before emergence, resulting in damping-off in pre-emergence, while Cercospora kikuchii (Tak. Matsumoto & Tomoy.) M.W. Gardner causes seed discoloration (Alloatti et al., 2015). In soybean (Glycine max L.), Pythium Nees causes a purple spot (Upchurch & Ramirez 2010) and, when inside the embryonic tissues, it causes necrosis of cotyledons and vascular elements (Pathan et al., 1989). The fungi Fusarium fujikuroi (as -Fusarium moniliforme), F. oxysporum and Penicillium spp. also cause seed rot (Debnath et al., 2012). flavus, Curvularia spp. and Cladosporium spp. caused total discoloration of the integument of stored melon seeds. In wheat, different species of the Fusarium genus caused loss of grain yield, stand reduction, and seed rot (Laram et al., 2020). On the other hand, Fusarium proliferatum in sesame caused damping-off, reducing seedling growth and vigor (Nayyar et al., 2018).
Seed-associated pathogens can also produce mycotoxins that cause diseases in humans and animals, feeding on seeds directly or indirectly (Karaca et al., 2017). Most of these mycotoxins are potentially carcinogenic, teratogenic, tremorogenic, nephrotoxic, immunotoxic or hemorrhagic, and cause fungal contamination in oilseeds, resulting in the presence of mycotoxins in the extracted oil (Bhat et al., 2015). Fungi of the genera Aspergillus, Fusarium and Penicillium are mycotoxin producers (Bhat et al., 2010).

Forms of disease control
Treatments to protect seeds from pests and diseases have been used for centuries. For example, in the year 1600, wheat seeds were already treated with salt to help control wheat rust (Hitaj et al., 2020). Historically, synthetic fungicides were developed using compounds containing sulfur, copper, and mercury (Mancini et al., 2013). Mercury-based treatments are Phenyl Mercury Acetate (PMA), Methoxyethyl Mercury Chloride (MEMC), Ethyl Mercury Chloride (EMC), Mercury Chloride, and Mercuric Eoxide. Non-mercurial treatments are Thiram, Captan, Carbendazim, Metalaxyl, copper carbonate, copper sulfate, and cuprous oxide (Kunta et al., 2020).
In recent years, different control methods have been used for seed treatment. According to Spadaro et al. (2017), there are other treatments for pathogen control in seeds besides the use of synthetic chemicals. Physical treatments include mechanical, thermal, ultrasonic, and radiations and use inorganic natural products, such as copper, phosphate, sulfur bicarbonates, clay, and potassium. There are also treatments with antagonistic microorganisms, such as filamentous fungi, yeasts, and bacteria as biocontrol agents as well as the use of resistance-inducing compounds, such as elicitors and natural organic compounds, such as plant extracts and essential oils.
In physical treatments, the use of hot water, hot air, and electrons are more frequent. In biological treatments, biological control agents (BCAs) are used, which include fungi and bacteria (Mancini et al., 2013). Jiao et al. (2016) proved that radio frequency (RF) heating assisted by hot air has the potential to inhibit fungi and ensure biochemical and physiological quality of grain seeds. Carvalho et al. (2011) studied the effect of Trichoderma harzianum Rifai isolates as biocontrol of pathogens in bean seeds (Phaseolus vulgares L.) and proved that these isolates are efficient to reduce incidence of Aspergillus, Cladosporium, and Sclerotinia sclerotiorum (Lib.). Lima et al. (2016) evaluated the effect of plant extracts and essential oils in the control of Alternaria alternata and A. dauci in Daucus carota L. seeds and observed that garlic extract and orange essential oil showed potential to control both pathogens.
However, both synthetic and natural chemical methods must meet some requirements for an effective seed treatment.
Treatments should be able to reduce the number or transmission rate of target pathogens to acceptable numbers without decreasing seed germination or vigor and storage capacity and be less toxic to humans, animals, and the environment . Treatment efficacy depends on the internal infestation degree of the seed, the amount of inoculum in the lot, specificity, and the treatment phytotoxic potential (Du Toit, 2004).

Are plant extracts effective for the control of fungal pathogens in seeds?
In this review, we evaluated 18 articles on the use and efficacy of plant extracts for the control of fungal pathogens in seeds of cultivated plants. The literature survey showed that 100% of the articles reviewed report that plant extracts were effective to control pathogens. However, there is a difference in the efficacy level between each extract. In addition, 63% of the studies reported an increase of seed germination, 21% reported no change in germination, 5% reported negative interference, and 11% did not evaluate the effects of plant extracts on seed germination. Among the extractors used to prepare Research, Society andDevelopment, v. 10, n. 14, e174101421846, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i14.21846 6 the extracts, 72% of the studies mentioned the use of aqueous extracts, 11% used ethanol, and 5.55% used citric, alcoholic, and citric acid + sodium benzoate + potassium sorbate and propylene glycol + water + sodium benzoate + potassium sorbate (Table   1). Borges et al. (2018) state that plants have in general secondary metabolites responsible for the synthesis of several bioactive substances, which limit growth of other plants and protect against insects and pathogens, showing thus efficacy in disease management. Plant extracts contain large amounts of these bioactive substances, such as alkaloids, cyanogenic glycosides, glucosinolates, lipids, phenolics, terpenes, polyacetylenes, polythiens, tannins, phenols, resins, volatile and fixed oils that are stored in specific plant structures, such as in leaves, bark, seeds, fruits, and roots (Gupta et al. 2012;Borges et al. 2018).
The methods to extract these compounds must be effective, providing good extraction yield and efficacy (Gupta et al., 2012). Many solvents are used for the extraction of these compounds, such as water, methanol, ethanol, ethyl acetate, and others; however, the right solvent should be chosen for each extraction to have the best results (Ong et al., 2021). Raw water or alcohol are extractors usually used to select plants with possible antimicrobial activity (Yazdani et al., 2011).
Plant extracts have a narrow range of specific action mode making them suitable for the control of specific pathogens.
These plant extracts also have limited persistence in the field and a shorter shelf life than synthetic chemicals; nevertheless, they do not pose a residual threat and can be used in integrated pest management (IPM) (Zaker, 2016). However, plant extracts have many different molecules in their composition, which vary depending on the plant origin and the extraction process. For example, "Neem" extract can be found more than 50 different molecules. Azadicachtine is important in pest management and is one of its main constituents (Alabouvette et al., 2006). Similarly, quercetin, ß sitosterol, and polyphenolic flavonoids are fundamental in the management of fungal diseases .
Degradation and volatilization of bioactive substances reduce efficacy of vegetable-based products under field conditions. However, an alternative to mitigate this disadvantage is to formulate bioactive vegetable products using biodegradable polymers, plasticizers, stabilizers, and antioxidants (Borges et al., 2018).
Despite the increased use of plant extracts as an alternative to synthetic chemical molecules, most studies did not compare the efficacy of extracts in replacement of fungicides (67%). However, 33% of the studies reported this comparison and stated that the extracts tested are promising to replace synthetic fungicides. Santos et al. (2020) used formaldehyde, mancozeb, and garlic extract in soursop seeds and found greater reduction in the incidence of Lasioplodia theobromae (Pat.) Griffon & Maubl. and Fusarium sp. with emphasis on garlic extract that controlled 100% of the fungus Fusarium sp. Sousa et al. (2018) treated pumpkin seeds with garlic extract Trichodel® and Captan® and verified that the extracts reduced the incidence level of Alternaria sp., Epicoccum sp., Fusarium sp., Nigrospora sp., Phoma sp. to less than 20%. Arefin et al. Thus, plant extracts show efficacy in the control of phytopathogenic fungi associated with seeds. However, more studies are needed to better understand the extraction methods, modes of action, maintenance, and chemical stability of these products, as well as their comparison with other control methods for various pathosystems, due to their specificity, to apply to other cultures and to use them on large scale.

Conclusion
Plant extracts are effective to control fungal pathogens in seeds, as reported in several studies. Plant extracts act directly or indirectly on pathogen growth in the seed due to their bioactive compounds. They have a narrow range of mode of action, making plant extracts suitable to control a specific pathogen. Thus, changes caused by the extracts are reflected in the severity reduction of pathogens in plant seeds; therefore, extracts can be considered a management tool for fungal pathogens that affect seeds. Additional studies are needed for a better understanding of these products to expand their use to other cultures and produce them on large scale.