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Background:
Systematic Review

Plant Protection Products to Control Alternaria Brown Spot Caused by Alternaria alternata in Citrus: A Systematic Review

by
Alfonso Garmendia
1,†,
María Ferriol
1,†,
Roberto Beltrán
2,
Francisco García-Breijo
2,
María Dolores Raigón
3,
María Del Carmen Parra
2 and
Hugo Merle
2,*
1
Instituto Agroforestal Mediterráneo, Universitat Politècnica de València, 46022 Valencia, Spain
2
Departamento de Ecosistemas Agroforestales, Universitat Politècnica de València, 46022 Valencia, Spain
3
Instituto de Conservación y Mejora de la Agrobiodiversidad Valenciana, Departamento de Química, Universitat Politècnica de València, 46022 Valencia, Spain
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(6), 1343; https://doi.org/10.3390/agronomy15061343
Submission received: 2 May 2025 / Revised: 28 May 2025 / Accepted: 29 May 2025 / Published: 30 May 2025
(This article belongs to the Section Pest and Disease Management)
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Review Reports Versions Notes

Abstract

Alternaria Brown Spot (ABS) is one of the most critical diseases affecting susceptible mandarins worldwide, being a limiting factor in their cultivation. Although there are numerous reports on effective plant protection products against the disease, field control is failing. In the literature, some of the results are contradictory, depending on the study and experimental scale. Therefore, this paper aimed to analyze the empirical evidence to answer the following questions: (i) What plant protection products have been used to control ABS? (ii) What are the methodologies used to test the substances? (iii) Why is ABS field control failing? An extensive literature search was performed in five databases: WoS, Scopus, Google Scholar, PubMed, and SciELO. The search string used was “Alternaria alternata” AND “Citrus”. Records were classified into ten groups according to their main topic. Group 3 “microorganisms and natural substances” and group 4 “fungicides” were full-text reviewed for data extraction (98 reports). Details of the microorganisms, natural substances, and fungicides used against A. alternata, as well as summaries of the methodologies, are provided. During this research, we highlighted significant aspects that may be hindering the control of Alternaria alternata in citrus: long periods of fruit sensitivity, abundance and floatability of inoculum, rapid infections, the appearance of resistance to fungicides, moderate effectiveness inhibiting the germination of conidia, uncertainty about the times of application, and persistence of the products.

1. Introduction

Alternaria Brown Spot (ABS), caused by the ascomycete fungus Alternaria alternata (Fr.) Keissler causes leaf, twig, and fruit lesions, reducing the yield and fruit quality of many tangerines (Citrus reticulata Blanco) and their hybrids [1]. Among the most affected cultivated varieties are cultivars from ‘Dancy’ as a direct or indirect parent, such as ‘Fortune’, Minneola’, and ‘Nova’ [2]. Several varieties of grapefruit, the mandarin ‘Emperor’, and the hybrids ‘Murcott’, Orlando’, ‘Fairchild’, and ‘Page’ are also affected [3]. The new variety ‘Leanri’ is seriously affected as well.
ABS is the most critical disease for susceptible tangerines worldwide, including all countries where these varieties are grown, such as Spain, Italy, the USA, Israel, China, and Brazil [4,5,6,7]. The disease represents a significant problem for susceptible varieties to the point that it constitutes a limiting factor for profitable cultivation. In fact, the appearance of ABS in Spanish citrus-growing regions has already forced the abandonment of ‘Fortune’ mandarin production, given the difficulty of controlling this disease using fungicides [8]. Other susceptible varieties, such as ‘Nova’, are beginning to be phased out of cultivation as current field treatments are failing (personal communication from farmers’ organizations).
Alternaria spp. have specific cells known as appressoria, which play an important role in recognizing the host through certain hydrophobic materials released from the host surface [9,10]. The pathogenicity of Alternaria alternata in citrus primarily depends on the biosynthesis of the host-selective ACT toxin, which is released during conidial germination [11]. This toxin compromises the integrity of host cell plasma membranes, ultimately leading to cell death, and subsequently spreads through the vascular system [12,13]. In addition to toxin production, the fungus secretes cell wall-degrading enzymes (CWDEs), which are also critical for successful infection of citrus tissues [14]. Within necrotic citrus tissue, A. alternata must detoxify reactive oxygen species (ROS) to survive in the oxidative environment and establish successful colonization [15].
Therefore, ABS is a contact disease, where highly buoyant conidia reach sensitive organs (young leaves and fruits), initiating an infection process [16]. Alternaria alternata survives for a long time in the soil or in the leaves as conidia [17], and infection of leaves in spring results in inoculum buildup that makes the disease difficult to control in fruits later in the season [18].
There are numerous reports on plant protection products and treatments used to control ABS; however, some results are contradictory throughout the studies and even between experimental scales (i.e., laboratory and field experiments). Considering these diverse results, a systematic review may help clarify this variability. While traditional reviews may fail in selecting those studies that argue for the authors’ initial viewpoints, systematic reviews are based on unbiased data extraction from a subset of studies that fit the pre-established eligibility criteria, aiming to provide a robust and sensible answer to a focused research question. Therefore, a systematic review is proposed for the first time to identify the most effective plant protection products and assess their potential to control ABS under field conditions.
Therefore, this paper aims to analyze the empirical evidence to answer the following questions: (i) What plant protection products have been used to control ABS? (ii) What are the methodologies used to test them? (iii) Why is ABS field control failing?

2. Materials and Methods

A systematic review uses explicit, systematic methods to collate and synthesize findings of studies that address a clearly formulated question [19]. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed [20]. This methodology was created by researchers from around the world to standardize and improve the validity of systematic reviews and meta-analyses [20]. The protocol has been published in the INPLASY registry (DOI number: 10.37766/inplasy2025.5.0011).

2.1. Step 1: Information Sources and Search Strategy

First, a broad literature search to obtain all records on the topic was carried out in five databases: WoS, Scopus, Google Scholar, PubMed, and SciELO. The databases were consulted in January 2025. The search string used in all databases was, “Alternaria alternata” AND “Citrus” and, whenever possible, it was limited to the title, abstract, and keywords (Table 1). Searches were not limited to publication dates (all years), document type (all), or language (all).

2.2. Step 2: Initial Classification of Records and First Data Collection

From the 891 identified records, duplicates were removed (Figure 1). The remaining 437 records were examined by title and abstract and classified into 10 groups (GRP) according to the main topic to which they referred. At this point, an initial reading of the abstract of each report was performed to obtain an overview of Alternaria alternata not only related to treatments to control the pathogen but also to taxonomy, phylogenetics, pathogenicity, etc.

2.3. Step 3: Eligibility Criteria for Full-Text Review

From the 10 groups, group 3 (microorganisms and natural substances; n = 59) and group 4 (fungicides to control ABS; n = 54) were selected for full-text screening (n = 113, Figure 1). These two groups included information on the plant protection products used to control A. alternata in both laboratory and field experiments, which are required to answer the research questions. Other groups, such as groups 6 and 8, are highly interesting in the development of other strategies, such as the breeding of new resistant varieties. This systematic review focused on available or candidate plant protection products against A. alternata in susceptible varieties currently being cultivated. Conferences (n = 4), reports with no outcome of interest (n = 7; remoteness of results from the main topic), reports focused on post-harvest disease and not on ABS (n = 2), and wrong species (n = 2; seeding albinism in lemon) were excluded (Figure 1), resulting in a total of 98 records.

2.4. Step 4: Charting Data

The data from the 98 records were included in two tables: one for microorganisms and natural substances (Table S1) and one for fungicides (Table S2) with the following items:
  • Article identifiers: authors, year of publication, country, and title.
  • Target species: plant species, variety, and disease (A. a. general, ABS, or post-harvest losses).
  • Substance information: group, common (commercial) name, scientific (substance) name, additional information.
  • Experiment information: type (I, II: when two different types of experiments were used in the same study), concentration, and additional information (field experiments: yes/no).
  • Main result: text (the results explained in the text), MIC (Minimum Inhibitory Concentration), MGI (Mycelial Growth Inhibition), EC50% (Concentration causing 50% growth inhibition), effectiveness (Type I, Type II).
  • Conclusion: text.
  • Interest: importance (goes from 1 to 5, and reflects the closeness to the main topic), reliability scale (goes from 1 to 3, low, medium, high; reflects the quality and reproducibility of experiments).

2.5. Step 5: Risk of Bias, Effect Measure, and Heterogeneity Among Study

The journals where the articles were published were analyzed. All journals were peer-reviewed by at least two independent reviewers. Reports from journals without peer review and quality criteria were not included. All included studies used standardized laboratory protocols. The types of experiments used in each case were summarized in a dedicated section “Methodologies used to test the antifungal activity”. No missing results were found. The effect measure varies depending on the type of experiment and even within the same experiment. Therefore, in the data extraction table (Tables S1 and S2), different columns were used for each type of measurement. Effect was categorized (low, medium, and high) for the overall interpretation of the results. The robustness of the synthesized results was expressed by the number of reports that tested each fungicide. Confidence in the body of evidence for a result was presented as a “reliability index” for each report. The results were heterogeneous. The main causes of heterogeneity were analyzed and summarized as follows: (i) in laboratory experiments: the use of different pathogen strains, the concentration of substances, and the development of resistance; (ii) in the field: plot characteristics, the initial amount of inoculum, weather conditions, and the number of applications.

2.6. Step 6: Collating, Summarizing, and Reporting the Results

A descriptive numerical summary of the characteristics of the included studies was prepared. Tables and graphs were created to reflect the overall number of studies included, study designs and settings, publication years, reported outcomes, and the countries where studies were conducted. All the statistical analyses were performed using R V4.3.3 [21] and RStudio 2023.06.1 [22]. For the graphics, ggplot2 V3.4.4 [23] and waffle V1.0.2 [24] packages were also used.

3. Results

3.1. Search in Databases

In total, 891 articles were retrieved from five databases. The WoS database contributed the majority of articles for this review, at 39% of the total (Figure 1). The Scopus, Google Scholar, PubMed, and SciELO databases represented, respectively, 31%, 16%, 12%, and 2% of the papers found. The databases with a broader search spectrum, such as WoS, Scopus, and Google Scholar, retrieved the largest number of records. Other databases were searched, but they only increased duplicates. Most records were duplicated twice or even 3–4 times, coming from the different databases. When duplicates were eliminated (n = 454), a total number of 437 records was obtained for the next step (Figure 1).

3.2. Initial Classification

After removing the duplicates, records were classified into 10 groups according to their content and main topic (Figure 2). At this abstract screening phase, any record that did not relate to the topic was rejected (n = 35; 8%). One of the most numerous report groups was group 8 (molecular characterization and diversity) with 63 articles (15.7%, Figure 2). Other significant groups were group 6 (metabolic pathways; 13.2%), group 5 (host–phytopathogen interactions and ecophysiology; 11.4%), and group 9 (toxins; 9.5%) with a large number of reports (Figure 2). Group 2 (A.a.. pathogen in citrus; 7.5%) included first reports of the disease and varieties’ sensibility studies. Abstracts were read from these groups, which contributed to obtaining an initial broad overview. Group 3 (microorganisms and natural substances; 14.7%) and group 4 (fungicides to control ABS; 13.4%) were not examined at this point as these records were moved to the next phase to continue the processes of screening, eligibility, and data extraction.

3.3. Studies Characteristics of Group 3: Microorganisms and Natural Substances

After applying the eligibility criteria, 56 reports were included in this group for full-text review. In those 56 papers, the antifungal activity against A. alternata was evaluated for more than 250 substances or microorganisms (Table S1). Studies were published from 1969 to 2024 (Figure 3A), with the main countries of publication being Brazil, USA, India, China, and Egypt (Figure 3B). The risk of bias was low across studies due to the high number of replicates and the use of standard experimental protocols.
There has been an increasing trend in the number of publications until 2018, with a decrease afterward (Figure 3A). Regarding this group, the reports could be grouped into 4 subgroups related to the nature of the treatments: microorganisms, essential oils, plant extracts, and other compounds (Figure 3C). The essential oil group was the largest with 22 articles (38%), followed by plant extracts (13; 25%), other compounds (12; 21%), and the smallest group, microorganisms, with 9 articles (16%) (Figure 4A). The heterogeneity among the study results could be due to the use of different strains of the pathogen and concentration of the substances.

3.4. Studies Characteristics of Group 4: Fungicides to Control ABS

A total of 42 papers were included in this group for data extraction and full-text review. In these 42 papers, the antifungal activity against A. alternata was studied at different experimental scales (from laboratory to field) for 38 fungicides and other substances (Table S2). Studies were published from 1996 to 2022 (Figure 4A), and the main countries of publication were the USA, Brazil, South Africa, Israel, and Spain (Figure 4B). Fungicides were grouped following the Fungicide Resistance Action Committee (FRAC) into 11 groups (Figure 4C). The most representative groups were Quinone outside Inhibitors (QoIs), copper (inorganic), dithiocarbamates, and DeMethylation Inhibitors (DMIs). Most experiments were conducted at a laboratory scale, but robust field experiments (with high number of replicates) were also performed (10 out of 42, Table S2). The confidence of each report was expressed using a reliability index ranging from 1 to 3. Most reports had medium–high reliability (2 and 3). For reports with low reliability (1), the results were thoroughly compared with other studies with higher reliability to analyze their consistency.

4. Discussion

4.1. Microorganisms and Natural Substances to Control ABS

The main objective of analyzing this group of reports was to answer: (i) what are the methodologies used to test plant protection products? (ii) which are the most effective natural substances or microorganisms to control ABS? and (iii) why they are not currently being used by farmers?
During this review, the effectiveness of the different plant protection products was easily compared within each study [25,26]. However, the effectiveness of the plant protection products was hard to compare among papers due to the use of different assays, concentrations, and modes of application and evaluation. In all cases, the antifungal activity depended on the applied concentration [27,28,29].

4.1.1. Methodologies Used to Test the Antifungal Activity of Microorganisms and Natural Substances

The type of experiment used to test the antifungal activity was related to the substance/treatment tested. For microorganisms, dual culture antagonist assays [30] and compartmentalized Petri dishes assays [31] were used, together with Mycelial Growth Assays (MGAs) in solid PDA medium and in vivo tests on fruits (where wounded fruits were usually inoculated and then treated) [32]. For essential oils, plant extracts, and other compounds, 7 different types of assays were used. The smallest experimental scale was represented by (1) micro-dilution method fungal growth assays (in liquid medium) used to calculate the MIC [33]. In addition, (2) MGA, (3) spore germination assays, and (4) pathogenicity tests on detached fruits were very common experiments throughout all the studies [34]. Those closer to field conditions were (5) detached leaves assays, (6) pathogenicity tests on seedlings, and finally, (7) field experiment under semi-real conditions [28,35,36]. Vapor contact assay (VCA) was only used in one report to evaluate volatile compounds of the essential oils [29].
Of all these techniques, the most frequent was MGA, which was used in 68% of the reports (Figure 5). For the evaluation of the antifungal effect in MGA, many studies used MGI, calculated as a percentage, (MGI (%) = [(Dc − Dt)/Dc] × 100), where Dc is the control diameter growth and Dt is the treatment diameter growth. However, in some other studies, the results were expressed as the concentration causing 50% MGI (EC50, mgL−1) [37] or as Mycelial growth rate (MGR) (millimeters of mycelial growth after several days) [38], which made results difficult to compare among all these studies.
The conidia germination assay (also called spore germination assay) and pathogenicity tests on fruits were frequent as well, having been used in 20% and 18% of the papers, respectively (Figure 5). The results of the germination assays were expressed directly as a percentage (number of conidia germinated over 100 conidia observed), as a percentage of inhibition (compared to the control), or even as the concentration that causes 50% inhibition of spore germination (EC50, mg/L) [37,39,40]. Results on fruits were evaluated as the diameter of the rot spot or, more commonly, as the area under the disease progress curve (AUDPC) and the percentage of protection [32].
Experiments closer to field conditions or the plant–pathogen interaction were the least used. Only two studies (3% of the total), one testing Cinnamomum zeylanicum Blume essential oil (EO) and the other one testing hexanoic acid, used field experiments, and only another two studies (in this group) used detached leaves assays [28,41]. Experiments with seedlings were used in 7 reports (12.5%), representing an interesting methodology between lab and field conditions [25,34,42,43].

4.1.2. Microorganisms to Control ABS

Approximately 9 out of 56 reports (16%) focused on the effect of microorganisms against A. alternata. Recombinant yeast Pichia pastoris (with cecropin A gene), P. guilliermondii, Saccharomyces cerevisiae, Trichoderma harzianum (strains 55 and IC-30), as well as the bacteria Pseudomonas syringae, P. flourescens (RK-1105), Burkholderia metallica (strain A53), B. territorii (strain A63), Bacillus subtilis (TV-6F and TV-17C), and Agrobacterium rubi (RK-33) showed more or less antifungal activity against A. alternata on MGAs [44,45,46,47]. For Burkholderia metallica and B. territorii, a strong in vitro antifungal activity against A. alternata was reported [48]. Trichoderma harzianum and several non-pathogenic bacterial strains significantly reduced disease severity on mandarin fruits during storage, all related to chitinase, glucanase, and protease enzyme activities [32,49].
Several studies pointed out that the use of these microorganisms requires a holistic approach, evaluating all potential side effects. In some cases, it can be difficult to distinguish between potentially useful isolates and those capable of causing plant diseases [28,50]. The microbiome associated with crop plants has a strong impact on their health and productivity [48], and the massive inoculation with just one microorganism could cause imbalances. Therefore, biocontrol with microorganisms requires a broad approach that takes into account complex ecological interactions, the production of metabolites (enzymes) and phytohormones, competition for space and nutrients, and the use of local strains adapted to the local climatic conditions [50,51].

4.1.3. Natural Substances to Control ABS

  • Essential Oils to Control ABS
The EO subgroup was the most widely studied with 22 out of 56 papers (39%). Over 100 EOs have been tested for their in vitro antifungal activity against A. alternata (Table S2). Most of the EOs had a partial inhibitory effect on the fungus, showing that the higher the concentration used in the culture media, the greater the direct fungi–toxicity effect on pathogen-inhibiting mycelial growth [28]. The EOs of Thymus vulgaris L., Cinnamomum verum J. Presl, Artemisia monosperma Delile, Origanum onites L., and Brassica nigra (L.) W. D. J. Koch stood out as the most effective ones [26,29,37,52]. For example, 0.6 mg/mL of T. vulgaris EO caused 70.8% of MGI [53], 0.054 mg/mL of A. monosperma EO caused 50% MGI [37], and 0.02 mg/mL of O. onites EO completely inhibited mycelial growth [26]. When the active ingredient of the EO was identified, less concentration of the pure active ingredient was necessary to have the same effect. Therefore, Thymus EO had a MIC of 500 µg/mL, while thymol had a MIC of 250 µg/mL [38]. Similarly, Cinnamomum zeylanicum EO had a MIC of 500 µg/mL, eugenol had a MIC of 250 µg/mL, and trans-cinnamaldehyde (a second active ingredient in the EO) had a MIC of 62.5 µg/mL [33]. In this last report, Nativo® commercial fungicide, composed of trifloxystrobin + tebuconazole (1:2 m/m) at 800 g L−1, was used as a control giving a MIC of 18.75 µg/mL. Other fungicides were used as controls, but the one that showed the lowest MIC was always Nativo® [33].
Citrus essential oils (CEOs) were also studied against A. alternata but with some contradictory results. While for Affes et al. [41] and Ajayi-Moses et al. [54], several CEOs from peels had a weak antifungal effect (with MICs over 12,000 µg/mL); for Azevedo et al. [28], EOs from mandarin peels had an effect high enough to be considered as an alternative method to control A. alternata [28,41,54]. No significant differences were found in the activity of EOs from ripe and unripe citrus fruit peels [28]. Although the studies could be compared, the minimum concentration to obtain 100% MGI was not calculated. To obtain 100% MGI, the EO of Citrus × sinensis (L.) Osbeck epicarp was applied at 0.5% [55] or at 0.2% [56]. C. × sinensis EO from fresh leaves was applied at 0.075% to obtain the same result [57]. The best result was obtained using the hydro-distillation of Citrus × aurantium L. mature leaves with 0.1 mg/mL for 100% MGI [58]. Regarding CEOs, several studies point to concentrations of around 2000 μg/mL to completely inhibit mycelial growth [59], which were usually higher than the concentrations indicated for Thymus, Artemisia, or Origanum EOs.
  • Plant extracts to control ABS
Approximately 13 papers out of 56 (23%) studied the effect of plant extracts against A. alternata, being the second-most numerous subgroup within group 3. The concentrations used to obtain antifungal activity were higher than those used with EOs. For example, Triaca et al. used concentrations of 20 and 40% in a PDA medium and only the 40% concentration of the fermented extract of Trifolium pratense L. was effective, while De Lima et al. used concentrations of 10, 20, and 30%, finding a moderated effect of Allium sativum L. extract at the maximum concentration; Citrus peel phenolic extracts from the variety ‘Mossambi’ were effective at 7% concentration [60,61,62]. When the active ingredients of the extracts were studied, the antifungal activity was mostly attributed to the phenol and sterol compounds [63,64]. Therefore, the best results for plant extracts were obtained with β-sitosterol and β-sitosteryl linoleates isolated from Anadenanthera colubrina (Vell.) Brenan, which had MICs of 250 and 500 µg/mL, respectively, against A. alternata [63]. Similar MICs were found for EOs, but still far away from Nativo® commercial fungicide with around 20 µg/mL MIC. The polyphenolic extract of Citrus × sinensis at the highest concentration assayed (1.5 gL−1) completely inhibited the conidial germination and growth of the fungal pathogen [64]. A moderated antifungal effect (MGI around 30–60%) was found for lemon by-product aqueous extracts [39] or Myrcia splendens (Sw.) DC. mature leaf extracts [26], although fermented extracts were generally more active than non-fermented ones [62]. On the other hand, Anadenanthera spp. Speg and Caesalpinia ferrea C. Mart. were highlighted as promising extracts to control A. alternata [25,27,42,43].
  • Other compounds to control ABS
Within this subgroup, substances as diverse as silver and vanadium nanoparticles, salicylic acid, chalcones, hexanoic acid, and chitosan films, among others, were included. Approximately 12 out of 56 (22%) papers were included here. Spraying citrus plants with 1 mM hexanoic acid four days before the first infection reduced the disease incidence, leading to smaller lesions (50% protection rate) and protection against A. alternata lasting for at least two months [8,65]. Among 137 chalcones, only chalcones D7 and D8 (B-ring as a 2,4,5-trimethoxyphenyl group) at 500 μg/mL showed moderate antifungal activity [34].
Vanadium and silver nanoparticles at 100 μg/mL showed strong antifungal activity [66,67], while nonspecific lipid transfer protein at 100 mg/mL only reduced spore germination of A. alternata to 51.6%, showing moderate antifungal activity [68]. The inhibitory activity of haloacylated cephalosporin TM1s against A. alternata was stronger than that of the positive control prochloraz [69]. Spraying salicylic acid showed an effect in protecting the treated fruits against fungal invasion throughout the 20 days of storage [36]. Finally, the evaluation demonstrated that CHI/AntiFun-LM films gained considerable antifungal properties against fungi responsible for post-harvest decay [70].

4.1.4. Summary of the Findings of the Biological Control and Natural Substances Group and Why These Plant Protection Products Are Rarely Used Under Field Conditions

Many substances and microorganisms showed moderate to high antifungal activity against A. alternata. For some of them, the effectiveness was comparable to that of the controls with fungicides, but still far from the effectiveness shown in a laboratory by some fungicide mix like trifloxystrobin + tebuconazole (20 µg/mL MIC). Trans-cinnamaldehyde, an active ingredient from Cinnamomum zeylanicum EO, had a very low MIC (62.5 µg/mL), even lower than most fungicides [33].
Thus, the vast majority of studies found effective protection products with antifungal activity, and in most of them, the substances were described as promising candidates to control ABS in citrus [8,27,29,49]. Nevertheless, after consulting many citrus grower associations in Spain, these plant protection products are not currently being used by citrus growers to control the disease under actual field conditions. Therefore, ABS in citrus remains an unsolved problem for susceptible varieties.
Several natural substances could replace fungicides, but this would require robust field experiments. Only 2 of the 56 papers conducted field experiments, and the results showed only partial protection against ABS. The lack of evidence of the effectiveness of these plant protection products in the field limits its use by farmers.

4.2. Fungicides to Control ABS

The objective of analyzing this group of reports was to find out which fungicides were the most effective for ABS control, what methodologies were used to test their effectiveness, and whether they were effective or not under field conditions. At the end of the section, we discussed the difficulties of field control, which would explain the lack of correspondence between laboratory and field experiment results. Results of experiments dealing with fungicides to control ABS were expressed in very dissimilar units, although most reports expressed results as percentages. In laboratory experiments, the percentage of MGI and the percentage of germination inhibition were common. In field experiments, the percentage of marketable fruit was usually used. Therefore, to be able to compare the results of the reports, we have used the percentage of inhibition or percentage of marketable fruit, and we have assigned them to low (<40%), medium (40–70%), and high (>70%) effectiveness.

4.2.1. Methodologies Used to Test the Antifungal Activity of Fungicides

The experiments performed to test the activity of the fungicides were similar to those used for the natural substances, albeit less diverse. At the laboratory scale, mycelial growth and spore germination assays were mostly used. In addition, at an intermediate scale between field and lab, detached leaves assays and seedling assays (in the greenhouse) were likewise used. Field experiments were performed more frequently and robustly (with larger sizes) than for natural substances, but they still represented a low percentage within the group (24%; only 10 of the 42 reports included field experiments).

4.2.2. Details of Fungicide Groups to Control ABS

  • Copper (inorganic) group
According to the Fungicide Resistance Action Committee (FRAC) Code List for 2024, copper molecules are chemicals with multi-site contact activity (MSCA), and therefore are generally considered a low-risk group without any signs of developing resistance to the fungicides [71]. Several copper substances such as Bordeaux mixture, copper hydroxide, copper oxychloride, cuprous oxide, and tribasic copper sulfate have been tested to control Alternaria alternata (Table 2). Effectiveness was variable depending on the formulation, type of experiment, and report.
Copper oxychloride was the most tested copper molecule (9 reports, Table 2). In lab experiments, effectiveness varied from low [72] to medium [73]. For some authors, copper oxychloride showed high effectiveness (89%) in the field experiments with 8 applications [76]. However, for some other authors, field effectiveness was medium (50–60%), with 10 applications [77], or even low [74]. Copper hydroxide was the second-most tested copper molecule (5 reports, Table 2). In lab experiments, effectiveness was low to medium and variable [72,81], while in field experiments, it showed medium effectiveness with 14 applications [82]. Copper hydroxide was also tested on seedlings with medium effectiveness (50%) and very low persistence (only two days) [96]. Both copper oxychloride and copper hydroxide were also tested by being mixed with oil, but showed no clear improvement in efficacy [74,82]. Bordeaux mixture and tribasic copper sulfate were very ineffective under lab conditions while cuprous oxide had high effectiveness [72]. Vincent et al. [79,80] tested these copper substances, finding good effectiveness and persistence.
  • Dithiocarbamates group and other MSCA
Dithiocarbamates are MSCA fungicides as well [71], and therefore considered a low-risk group for resistance development. Mancozeb, Propineb, Maneb, Ferbam, and Metiram were tested to control A. alternata (Table 2). Mancozeb was the most frequently tested dithiocarbamate (8 reports, Table 2) and is also one of the most widely used fungicides to control ABS by farmers (in those countries where it is permitted). Mancozeb was highly effective in lab experiments with an inhibition percentage of around 70% [72,73]. Furthermore, it showed high effectiveness in inhibiting spore germination [72], which was not very common among other fungicides. In field experiments, the effectiveness was medium to high [75,79,82]. However, Peres and Timmer [77] had to perform 10 applications to obtain 60% of marketable fruits. Mancozeb was also evaluated in mixtures with other fungicides, obtaining medium to high effectiveness [74,84].
Propineb was tested in laboratory experiments and was found to be highly effective (around 85%), even more so than Mancozeb [73,85]. In the field experiments, it was tested in a mixture with copper and trifloxys, revealing medium to high effectiveness [74]. Maneb was ineffective in field experiments [84], while in the same study, Metiram showed medium effectiveness. Ferbam was ineffective in detached leaves assays [86].
Other MSCA fungicides tested were chlorothalonil (Chloronitriles) and captan (Phthalimides), with some contradictory results (Table 2). Chlorothalonil was ineffective in field experiments [84], but effective when mixed with pyrimthanil [82]. Captan in field experiments was effective for Miles et al. [82], but ineffective for Solel et al. [84].
  • DeMethylation Inhibitors (DMIs) imidazoles and triazoles group
DeMethylation Inhibitors belong to the “G” group according to FRAC: they affect sterol biosynthesis in membranes and are considered to be a medium-risk group for resistance development [71]. Prochloraz showed high effectiveness in mycelial growth assays with an MGI of 100% [73], while it showed low effectiveness in field experiments [84]. Tebuconazole showed medium to high effectiveness in lab experiments [81], but low effectiveness in the field [84]. Tebuconazole’s effectiveness was greatly improved when mixed with other fungicides such as trifloxystrobin [87]. Difenoconazole showed low effectiveness in the field [74,79], while pyrifenox was effective in mycelial growth assays [85] (Table 2).
  • Quinone outside Inhibitor (QoI) group
QoI was one of the most numerous groups since it was studied in many reports (Table 2). Resistance is known in various fungal species with target site mutations and, therefore, is considered a high-risk group for resistance emergence [71]. Azoxystrobin and pyraclostrobin have been widely tested, while trifloxystrobin, methoxycrylate, and famoxadone have been tested to a lesser extent (Table 2). Azoxystrobin was found to be ineffective [88], highly effective [85], or showed low to medium effectiveness in laboratory experiments [72,81]. These contradictory results were probably related to the variable resistance degree of the strains used. Jamiołkowska [89] described its effectiveness as medium but of short duration (the effect lasted only a few days). In field experiments, it showed medium effectiveness [74,82], but 10 to 14 applications were required. Numerous studies have reported the appearance of resistance, rapid laboratory-emergent resistance, and cross-resistance [90,91,92,93,94].
Pyraclostrobin obtained better results than Azoxystrobin, although resistance was also detected. In laboratory experiments, it showed high MGI but slight inhibition of spore germination [88,95]. Pyraclostrobin was highly effective in seedling experiments, although its effect lasted only 5 days [18,96]. In field experiments, it showed high effectiveness [74,82] even with 8 applications [76]. Resistance and cross-resistance were widely identified for pyraclostrobin [83,90,91,93,94].
Trifloxystrobin showed low effectiveness in inhibiting conidia germination [81]. In field experiments, it was effective for Colturato et al. [74], but not for Miles et al. [82]. In this same study, methoxycrylate was more effective in the field than trifloxystrobin [82]. Famoxadone showed medium to low effectiveness and only two days of persistence in experiments with seedlings [96].
  • Benzimidazoles, Diarylamine, and Dicarboximides group
Carbendazim and Thiophanate methyl (Methyl Benzimidazole Carbamates, MBCs) were found to be not very effective in the lab [72,73]. They showed a positive effect in preventing fruit drop, but were not specifically tested for A. alternata in the field [75]. In addition, they are considered high-risk groups for resistance development [71].
Fluazinam, a diarylamine with low resistance risk, showed high effectiveness in the laboratory [72,97], but contradictory results were obtained in field experiments. For Highland and Timmer [98], it was effective in the field experiments, while for Solel et al. [84], it was ineffective in the field.
Iprodione and procymidone belong to the dicarboximides, a group of fungicides with a medium to high risk of resistance [71]. Iprodione showed high effectiveness both in the laboratory [95] and in the field [82,84,95]. However, resistance has already been detected and has even emerged rapidly in the laboratory [74,99,100]. Procymidone showed low effectiveness in field experiments [84].
  • Succinate-dehydrogenase inhibitor (SDHI) group
The SDHI group has also been important since several fungicides have been tested within this group. It is considered a medium- to high-risk group for the emergence of resistance [71]. Fluopyram, Flutolanil, and Thifluzamide were highly effective in laboratory experiments, but there is no data on their effectiveness in the field [81,97]. Boscalid showed medium effectiveness in Mycelial Growth Assays but was ineffective in inhibiting spore germination [95,97,101]. The emergence of resistance has already been described for Boscalid [101].
  • Others (not classified in previous groups)
Along with fungicides, other substances and techniques were evaluated to compare their effectiveness. Some were presented as “host plant defense inductors” or “plan activators”. However, in most cases, the mechanism of action was not clear. Laboratory experiments were promising for some plant protection products, such as natamycin (bio-fungicide) [74] or metallothionein [102], but no field experiments were carried out. The metallothionein mode of action is thought to be through zinc sequestration. As Alternaria alternata requires zinc to produce the mycotoxin, if zinc is not available, the toxin is not produced and there is no infection [102].
For many other substances, such as silicon [103,104], calcium nitrate [75,105], chitosan [76], salicyl-hydroxamic acid [83], or acibenzolar [18,82], the effectiveness was below that of fungicides. These substances were proposed as possible enhancers of fungicidal applications (in mixtures), but not as clear substitutes. The use of potassium phosphite stands out. In laboratory experiments, potassium phosphite did not show any antifungal activity; but unexpectedly, in field experiments, it showed effectiveness equivalent to that of fungicides (60–70%) [95]. The potassium phosphite effect was attributed to plant activation activity, but there was no clear evidence of its mode of action [95], and other mechanisms cannot be ruled out.

4.2.3. Summary of the Findings of the Fungicides Group to Control ABS and Why They Are Currently Failing to Control the Disease in the Field

Of the reviewed fungicides, mancozeb (already banned in several countries), pyraclostrobin, and iprodione were the most effective ones. However, Peres and Timmer [77] had to apply mancozeb 10 times to obtain 60% of marketable fruits, and evidence of resistance development has been provided for pyraclostrobin and iprodione [90,99]. The mixture of trifloxystrobin + tebuconazole was very effective as a control in the group of natural substances (Nativo®) [33] and with good field effectiveness within the fungicide group [87]. However, these two fungicides have shown moderate effectiveness when used separately.
Although highly effective fungicides were reported in the laboratory experiments, field control of A. alternata is currently failing in Spain and many other countries (data from cooperatives and other producer organizations). Results of this review showed that average results in field experiments were around 60–70% effective with 10, 14, or even 17 applications. This high number of applications with fungicides can be risky, harmful to the environment, and not suitable from an economic point of view. This is especially considering that most exporters will not harvest plots with 30% of infected fruits, since this means great losses due to damage in storage.
The main difficulties for field disease control can be highlighted. Several studies have shown that fruits are sensitive to infections from petal fall until a few days before harvest [106]. This means a very long period of fruit sensitivity of around seven to eight months. In addition, inoculum was found to be abundant in affected fields, with a high incidence of latent infections and highly buoyant conidia [16,81]. In fact, 86% of sampled flowers had latent infections [81]. Regarding the floatability of conidia, Badal et al. [16] observed that the concentration of conidia in the air followed a marked circadian periodicity, sometimes with up to 450 conidia/m3 at midday. Other authors observed that disease symptoms (brown and black spots) may appear up to 24 h after infection if fungal growth conditions are optimal [107].
All this evidence together—a long period of fruit sensitivity, an abundant inoculum with high buoyancy, and rapid infections—makes the field control of the disease extremely difficult. In addition, attempts to eradicate the pathogen in plots have failed: up to 20 applications with mancozeb and plots with 14–17 fungicide sprays resulted in a continuous inoculum buildup (data from the reviewed reports and producer organizations). In this regard, it is important to highlight that most fungicides inhibited fungal growth in the lab, but not conidia germination to the same extent [72,81,88,90,95]. Germination inhibition percentages were modest, probably indicating a more fungistatic than fungicidal activity, which may be behind the difficulty of eradication.
We have also found controversy regarding application periods, persistence, and rain fastness. For Solel et al. [84], the best application time was spring, and autumn applications were not effective; for Yogev et al. [95] and Vicent et al. [79,80], the autumn applications were very effective. Several weather-based models have been developed for timing fungicide sprays, based on temperatures, rain, and leaf wetness [16,108]. However, for Peres and Timmer [77], the use of the weather-based model did not improve fruit quality when compared to the scheduled program. Regarding persistence, for Mondal et al. [96], copper hydroxide and famoxadone provided 50% control of disease but for only two days after application and there was little or no disease control when the products were applied four or more days before inoculation. Only pyraclostrobin had a slightly better result, with five days of protection [96]. Low persistence and rain washout were mentioned by several authors, except for Vicent et al. [79,80], who reported a persistence of 28 days for several coppers and resistance to washout in a rain simulator.

4.3. Breeding for Disease Resistance

Beyond chemical and technical management strategies, breeding for disease-resistant citrus varieties offers also a promising approach to control Alternaria Brown Spot (ABS). This strategy requires a systematic review by itself due to the large number of reports, which was not the focus of this review. The main information during the classification phase was that proteomic analyses comparing susceptible and resistant citrus genotypes have revealed differential protein expression following infection. Resistant varieties exhibit elevated levels of proteins involved in ROS metabolism and immune responses, suggesting these factors play a key role in resistance mechanisms [109]. Furthermore, susceptibility to A. alternata has been associated with mitochondrial RNA processing. Disruptions in this process can sensitize citrus plants to the ACT toxin, exacerbating disease symptoms [110].

5. Conclusions

This systematic review aimed to collate and analyze all available empirical evidence on plant protection products used to control ABS caused by Alternaria alternata. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) methodology was used, with an initial broad literature search, classification of reports, eligibility criteria, and data extraction and analysis. The details of the microorganisms, natural substances, and fungicides used against A. alternata and a summary of the methodologies used to test them have been provided.
The possibilities of disease control with the use of synthetic fungicides are based on the strong antifungal effect of several fungicides (such as mancozeb, pyraclostrobin, and iprodione). However, control at the field level is limited by several factors, such as a long fruit sensitivity period, abundant inoculum in the plots and its high buoyancy, rapid infections, the appearance of resistance to fungicides, moderate effectiveness inhibiting the germination of conidia, low persistence, and rain washout. Therefore, synthetic fungicides control of ABS is failing, and it would be necessary to develop new formulations that improve aspects such as persistence or combine some other modes of action in the infection process beyond antifungal activity. With the current formulations, evidence shows that farmers will need many applications to achieve partial control of the disease.
The possibilities offered by microorganisms and natural substances are promising but require further development. There are microorganisms (such as Burkholderia spp.) and natural substances (such as thymol or trans-cinnamaldehyde) that are highly effective against A. alternata and are, therefore, good candidates for its control. However, most studies have been conducted at the laboratory scale. Field experiments and the development of formulations that allow their application under real field conditions are needed.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy15061343/s1. Table S1: Database for the extraction of information in the group of microorganisms and natural substances; Table S2: Database for the extraction of information in the group of fungicides.

Author Contributions

Conceptualization, H.M., A.G., M.F. and R.B.; methodology, H.M., M.D.C.P. and R.B.; software, A.G.; validation, H.M., M.F., M.D.R. and A.G.; formal analysis, H.M., A.G. and M.F.; investigation, H.M.; resources, H.M. and F.G.-B.; data curation, H.M., M.F. and A.G.; writing—original draft preparation, H.M.; writing—review and editing, H.M., M.F., A.G., R.B., F.G.-B., M.D.R. and M.D.C.P.; visualization, H.M., A.G., F.G.-B., M.D.C.P. and M.F.; supervision, H.M. and M.D.R.; project administration, H.M.; funding acquisition, H.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Citrusalte SAT, Artan Frut Coop. V., Casa Blava SAT 58CV, and Versol SAT 108 CV as part of a project undertaken with the Universitat Politècnica de València (Spain, UPV 20250023), of which Merle H. was the principal researcher. There was no additional external funding received for this study.

Data Availability Statement

The data presented in this study are openly available in DOI: 10.5281/zenodo.15146186 web: https://www.doi.org/10.5281/zenodo.15146186 web: https://zenodo.org/records/15146186, accessed on 4 April 2025.

Acknowledgments

The authors thank Carlos Zornoza for providing technical assistance and facilitating contacts with the farmers’ organizations.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
ABSAlternaria Brown Spot
ACTAlternaria Citri Toxin
AUDPCArea under the disease progress curve
CEOCitrus essential oil
DMIDeMethylation Inhibitor
EC50Concentration causing 50% growth inhibition
EOEssential oil
FRACFungicide Resistance Action Committee
MBCMethyl Benzimidazole Carbamate
MGAMycelial Growth Assay
MGIMycelial Growth Inhibition
MGRMycelial growth rate
MICMinimum Inhibitory Concentration
MSCAMulti-site contact activity
QoIQuinone outside Inhibitor
SDHISuccinate-dehydrogenase inhibitor

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Agronomy 15 01343 g001
Figure 1. PRISMA flow diagram for the article selection process. Papers were collected considering the search string (step 1). GRP = Groups. The classification are as follows: A. sp. in other species (GRP1), A. a. pathogen in citrus (GRP2), microorganisms and natural substances (GRP3), fungicides to control ABS (GRP4), host–phytopathogen interactions and ecophysiology (GRP5), metabolic pathways (GRP6), methodology (GRP7), molecular characterization and diversity (GRP8), toxins (GRP9), and hotchpotch (GRP10).
Figure 1. PRISMA flow diagram for the article selection process. Papers were collected considering the search string (step 1). GRP = Groups. The classification are as follows: A. sp. in other species (GRP1), A. a. pathogen in citrus (GRP2), microorganisms and natural substances (GRP3), fungicides to control ABS (GRP4), host–phytopathogen interactions and ecophysiology (GRP5), metabolic pathways (GRP6), methodology (GRP7), molecular characterization and diversity (GRP8), toxins (GRP9), and hotchpotch (GRP10).
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Figure 2. Classification of the records in 10 groups according to the main topic related to A. alternate (ordered from most to least abundant).
Figure 2. Classification of the records in 10 groups according to the main topic related to A. alternate (ordered from most to least abundant).
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Agronomy 15 01343 g003
Figure 3. Microorganisms and natural substances. Number of publications per year (A), per country (B), and subgroups inside the group (C).
Figure 3. Microorganisms and natural substances. Number of publications per year (A), per country (B), and subgroups inside the group (C).
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Figure 4. Fungicides. Number of publications per year (A), per country (B), and subgroups inside the fungicides group (C).
Figure 4. Fungicides. Number of publications per year (A), per country (B), and subgroups inside the fungicides group (C).
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Agronomy 15 01343 g005
Figure 5. Types of assays: MGA (68%), SGA (20%), DFA (18%), MMG (12%), SAA (12%), DCA (7%), DLA (4%), FEE (4%), DDM (4%), CPD (2%), VCA (2%). MGA (mycelial growth assay), SGA (spore germination assay), DFA (detached fruits assay), MMG (micro-dilution method fungal growth assay), SAA (seedlings assay), DCA (dual culture antagonist assay), DLA (detached leaves assay), FEE (field experiment), DDM (disk diffusion method), CPD (compartmentalized Petri dishes assay), VCA (vapor contact assay). Only one type of experiment by article was taken into account. Percentages were calculated by the total number of papers.
Figure 5. Types of assays: MGA (68%), SGA (20%), DFA (18%), MMG (12%), SAA (12%), DCA (7%), DLA (4%), FEE (4%), DDM (4%), CPD (2%), VCA (2%). MGA (mycelial growth assay), SGA (spore germination assay), DFA (detached fruits assay), MMG (micro-dilution method fungal growth assay), SAA (seedlings assay), DCA (dual culture antagonist assay), DLA (detached leaves assay), FEE (field experiment), DDM (disk diffusion method), CPD (compartmentalized Petri dishes assay), VCA (vapor contact assay). Only one type of experiment by article was taken into account. Percentages were calculated by the total number of papers.
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Table 1. Electronic search strategy.
Table 1. Electronic search strategy.
Database Specific Search StringPublishedDoc TypeLangn
WoS(TI = (Alternaria alternata AND citrus)) OR AB = (Alternaria alternata AND citrus) OR AK = (Alternaria alternata AND citrus) OR KP = (Alternaria alternata AND citrus)all yearsallAuto 343
ScopusTITLE-ABS-KEY (Alternaria AND alternata AND citrus)all yearsallall276
Google Scholarallintitle: Alternaria alternata citrus all yearsallall144
PubMed(Alternaria alternata [Title/Abstract]) AND citrus [Title/Abstract]all yearsallall109
SciELOAll indexes: (Alternaria alternata) AND (citrus)all yearsallall19
TI = Title; AB = Abstract; AK = Author Keywords; KP = Keyword Plus; ABS = Abstract; KEY = Keywords; Lang, the search language; n, the number of publications found.
Table 2. Fungicide group, with FRAC groups and code, substance name, resistance development, laboratory effectiveness, field effectiveness, number of references, and references.
Table 2. Fungicide group, with FRAC groups and code, substance name, resistance development, laboratory effectiveness, field effectiveness, number of references, and references.
GroupFRAC Code Substance ResistLab EffectivenessField EffectivenessRefsReferences
Copper (inorganic)MSCACopper oxychlorideNolow to medium low to high 9[72,73,74,75,76,77,78,79,80]
Copper hydroxideNolow to medium medium5[72,79,81,82,83]
Bordeaux mixtureNoineffectivenot tested3[72,79,80]
Cuprous oxideNohigh not tested3[72,79,80]
Tribasic copper sulfateNolownot tested1[72]
Dithiocarbamates MSCAMancozebNohigh medium to high 8[72,73,74,75,77,79,82,84]
PropinebNohigh medium to high 3[73,74,85]
ManebNonot testedineffective1[84]
FerbamNoineffectivenot tested1[86]
MetiramNonot testedmedium1[84]
ChloronitrilesMSCAChlorothalonilNonot testedineffective2[82,84]
PhthalimidesMSCACaptanNonot testedcontradictory 2[82,84]
DeMethylation Inhibitors DMIsProchloraz high low2[73,84]
Tebuconazole medium to high low3[81,84,87]
Difenoconazole not testedlow2[74,79]
Pyrifenox low to highnot tested1[85]
Quinone outside Inhibitors QoIsAzoxystrobinYescontradictory medium 12[72,74,81,82,85,88,89,90,91,92,93,94]
PyraclostrobinYeshigh high 12[18,74,76,82,83,88,90,91,93,94,95,96]
TrifloxystrobinYeslowcontradictory 3[74,81,82]
MethoxycrylateYesnot testedmedium 1[82]
FamoxadoneYesnot testednot tested1[96]
BenzimidazoleMBCCarbendazimYeslow not tested2[72,75]
Thiophanate methylYeslow not tested2[72,73]
Diarylamine FluazinamNohigh contradictory 4[72,84,97,98]
Dicarboximides IprodioneYeshigh high 6[74,82,84,95,99,100]
ProcymidoneYesnot testedlow1[84]
Succinate-dehydrogenase inhibitors SDHIsFluopyramYeshigh not tested1[81]
FlutolanilYeshigh not tested1[97]
ThifluzamideYeshigh not tested1[97]
BoscalidYesmedium to lownot tested3[95,97,101]
Others NatamycinNohigh not tested1[74]
Metallothionein Nohigh not tested1[102]
SiliconNomedium to lownot tested2[103,104]
Calcium nitrateNonot testedmedium to low2[75,105]
ChitosanNonot testedmedium to low1[76]
Salicyl-hydroxamic acidNomedium to lownot tested1[83]
AcibenzolarNomedium to lownot tested2[18,82]
Potassium Phosphite Noineffectivemedium to high 1[95]
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Garmendia, A.; Ferriol, M.; Beltrán, R.; García-Breijo, F.; Raigón, M.D.; Parra, M.D.C.; Merle, H. Plant Protection Products to Control Alternaria Brown Spot Caused by Alternaria alternata in Citrus: A Systematic Review. Agronomy 2025, 15, 1343. https://doi.org/10.3390/agronomy15061343

AMA Style

Garmendia A, Ferriol M, Beltrán R, García-Breijo F, Raigón MD, Parra MDC, Merle H. Plant Protection Products to Control Alternaria Brown Spot Caused by Alternaria alternata in Citrus: A Systematic Review. Agronomy. 2025; 15(6):1343. https://doi.org/10.3390/agronomy15061343

Chicago/Turabian Style

Garmendia, Alfonso, María Ferriol, Roberto Beltrán, Francisco García-Breijo, María Dolores Raigón, María Del Carmen Parra, and Hugo Merle. 2025. "Plant Protection Products to Control Alternaria Brown Spot Caused by Alternaria alternata in Citrus: A Systematic Review" Agronomy 15, no. 6: 1343. https://doi.org/10.3390/agronomy15061343

APA Style

Garmendia, A., Ferriol, M., Beltrán, R., García-Breijo, F., Raigón, M. D., Parra, M. D. C., & Merle, H. (2025). Plant Protection Products to Control Alternaria Brown Spot Caused by Alternaria alternata in Citrus: A Systematic Review. Agronomy, 15(6), 1343. https://doi.org/10.3390/agronomy15061343

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