First Occurrence of Coffee (Coffea arabica L.) Wilt Disease Caused by Neocosmospora falciformis in Saudi Arabia as Corroborated by Molecular Characterization and Pathogenicity Test

1  Introduction

The two primary species Coffea canephora (Robusta coffee) and Coffea arabica L. (Arabica coffee) produce coffee accounting for 70% and 30% of the world’s total commercial output of coffee, respectively. For the past four or five centuries, Yemen and Saudi Arabia have grown Arabica coffee on slender valleys and terraced mountain slopes at elevations reaching from 1200 to 1800 m [1]. In the southwestern regions of Saudi Arabia, including Al-Baha, Asir, and Jazan there are historic coffee plantations where some trees are more than 100 years old. In the southwestern area of Saudi Arabia, coffee plays a major role in the economy and is the primary cash crop grown by smallholder farmers, with social, and economic advantages for farmers in the mountainous areas. To increase interest in coffee production, research on coffee cultivation in the region has intensified [2]. Saudi Vision 2030 emphasizes transformative progress across various economic sectors, with the coffee industry being a key focus. In 2021, the coffee market was valued at USD 1.58 billion and is projected to grow to USD 2.22 billion by 2028 [3]. By 2026, Saudi Arabia aims to cultivate 1.2 million coffee trees as part of its agricultural goals. To support this initiative, the Ministry of Environment, Water, and Agriculture has introduced multiple programs, such as the Sustainable Agricultural Rural Development Program (REF), designed to promote and enhance coffee farming across the Kingdom [4]. The Saudi Coffee Company plans to invest approximately $320 million over the next decade to boost the coffee industry. This initiative aims to elevate annual coffee production from 300 to 2500 tonnes, which is expected to create numerous employment opportunities and drive sectoral growth [5].

Coffee farmers face numerous obstacles in their agricultural yields throughout tropical regions [6]. Climate change has affected coffee production both directly, through reduced crop yields and quality, and indirectly, by fostering the spread of fungal diseases and invasive pests [6–9]. The three main diseases that affect coffee trees are Coffee Leaf Rust (CLR) [10], Coffee Wilt Disease (CWD) [11,12], and Coffee Berry Disease (CBD) [7], which are triggered by Gibberella xylarioides, Colletotrichum spp., and Hemileia vastatrix, respectively. CWD has been mainly caused by Fusarium xylarioides, a soilborne fungus that induces vascular wilt and kills coffee plants [12]. However, Neocosmospora falciformis (formerly known as; Fusarium falciforme) and other species, for example, F. solani, F. lateritium, F. xylarioides, F. stilboides have been documented to cause wilt and dry rot to coffee worldwide [11–15]. Neocosmospora (Hypocreales, Nectriaceae), which was recently separated from the F. solani species complex (FSSC), is a genus of ubiquitous fungi with a global distribution that can be found in soil, water, air, living plant material, and plant detritus [16]. Neocosmospora is recognized as an emerging threat to coffee crops, particularly in Saudi Arabia and potentially on a global scale. However, there is limited understanding regarding the fungus’s distribution, its ability to cause disease, or the frequency of associated outbreaks.

Significant advancements in the taxonomy and phylogeny of F. oxysporum have been achieved through the application of DNA-based methods, including AFLPs, RFLPs, and RAPDs [17]. AFLPs, RFLPs, and RAPDs are also affected by significant homoplasy, fail to identify alleles, and the resulting groups are influenced by the quantity and variety of restriction enzymes, probes, and primers employed. RAPD is also characterized by low reproducibility [17]. Consequently, DNA markers are recognized as a reliable solution for addressing challenges related to the taxonomy and phylogenetic classification of Fusarium species [18–20]. DNA markers that have been used for delineating Fusarium and Neocosmospora species are the internal transcribed spacer (ITS) areas, such as the 5.8S gene, both the large (LSU; 28S) and small (SSU, 18S) subunits of the nuclear ribosomal RNA genes, along with protein-encoding genes, like the largest subunit of RNA polymerase II (RPB1), the second largest subunit of RNA polymerase II (RPB2), cytochrome oxidase subunit 1 (COX1, COI), β-tubulin (TUB2), translational elongation factor 1-α (TEF1-α), γ-actin and calmodulin (CaM) [20]. Unfortunately, none of the ribosomal markers have been proven to distinguish Fusarium and Neocosmospora at the species level. The reported markers that have a great species identification are TEF1-α and RPB2 [21]. EF-1α is frequently utilized as the primary identification marker due to its exceptional resolution capabilities for the majority of species, whereas RPB2 provides improved differentiation among closely related species [21]. Therefore, the implementation of multi-locus phylogenetic analyses is essential to support the separation of F. solani and its closely related species from Fusarium to the genus Neocosmospora [18].

The occurrence of coffee plant death due to a lack of necessary agricultural practices and phytosanitary measures is alarming, especially in the context of fungal diseases, indicating that CWD poses a significant obstacle for smallholder coffee farmers. The recent rise in CDW infections underscores the necessity for investigating Neocosmospora wilt as a developing economically significant disease of coffee based on field observations. Considering the recently reported coffee pathogens in Saudi Arabia [7–9,22], very little information regarding phytopathogens, insects, and other pathogenic agents has been documented in Saudi Arabia’s coffee. In light of this, the existing investigation represents the first attempt to characterize the possible fungal species related to wilt and decline diseases that may lead to probable losses of coffee in Saudi Arabia. The purpose of the existing investigation was to identify the fungi related to wilt-like symptoms in coffee based on phylogenetic, morphological, and pathogenicity assessment throughout in vitro and in vivo experiments.

2  Materials and Methods

2.1 Sampling and Isolation

Samplings were conducted from severely wilted coffee trees during August and September 2023 in different geographical regions in Saudi Arabia namely; Jazan, El Baha, Najran, and Asir regions. A total of 28 root samples from different trees manifesting wilt symptoms were collected. Isolation was done on Potato Dextrose Agar (PDA) medium amended with Ampicillin sulfate (0.1 g/L−1). The infected roots were surface-sterilized by immersing them for 5 min in a solution of 2% sodium hypochlorite, afterward washed three consecutive times with sterilized distilled water. They were then blotted using sterilized filter paper. The sterilized roots were sectioned into small pieces, each about with a diameter of 0.5 cm, and transferred onto a medium of Potato Dextrose Agar (PDA). Four fragments were positioned on every plate and maintained at 25°C for a span of 5 to 7 days. Colonies similar to Fusarium were sub-cultured and distinguished by molecular and morphological methods. The colonies growing on PDA were preliminarily identified as Fusarium, based on their depressed beige mycelia, concentric ring growth, and lack of sporodochia [23]. The primary evaluated characteristics encompassed microscopic features (including the existence of macro- and microconidia, along with chlamydospores) and macroscopic traits (including the presence of aerial mycelium, as well as the color and appearance of the colony). As noted by Stefańczyk and Sobkowiak [24], conidia were germinated for 1–2 days at 16°C on a water agar medium to obtain single-spored subcultures from the colony margin.

2.2 Molecular Characterization

2.2.1 DNA Extraction, PCR Amplification and Sequencing

From fresh mycelium of 7-day-old fusarioid isolates, the total genomic DNA was extracted employing the Dellaporta procedure [25]. DNA markers, including translational elongation factor 1-α (TEF1-α), the second-largest subunit of RNA polymerase II (RPB2), calmodulin (CaM), and the internal transcribed spacer (ITS) region, were employed. The primer pairs enumerated in Table 1 were employed for the amplification and sequencing of the respective gene areas. The PCR mixtures, as detailed by Alhudaib et al. [7], were prepared in a total volume of 25 μL. PCR was done utilizing a 2720 Thermal Cycler (Applied Biosystems, Foster City, CA, USA), and the amplification conditions for TEF1-α, CaM, and RPB2 were carried out according to the program of Khuna et al. [26], and for ITS by the procedure of Alhudaib et al. [7]. Sequencing and purification of the PCR products were done in forward and reverse directions at Macrogene Inc. (Seoul, Korea), following the manufacturer’s protocol. Utilizing MEGA v.7, the generated sequences were assembled and manually edited [27]. The sequence homology was assessed utilizing BLAST® vs. the sequence database of NCBI (National Center for Biotechnology Information, GenBank) (www.ncbi.nlm.nih.gov/genbank) (accessed on 01 February 2024).

2.2.2 Phylogenetic Analysis

The phylogenetic position of the Saudi Arabian collection was determined by combining and analyzing concatenated sequences of CaM, ITS, RPB2, and TEF1 with the reference sequences obtained from GenBank (Table 2). MAFFT [33] was employed to compute the multi-alignment of the sequences. MEGA XI v.11.0.8 [34] was employed for concatenating and trimming the multi-sequence alignment. The UFBoot [35] ML analysis was done with IQ-Tree 2.2.2.6 [36], employing the optimal model selected through ModelFinder [37], for each partitioned locus [38].

2.3 Pathogenicity Tests

Pathogenicity test was carried out on six-month-old coffee (Coffea arabica) seedlings following the previously published method [39]. Representative isolates (KSA 24-1, KSA 24-4, KSA 24-6, and KSA 24-14) of N. falciformis were selected and cultivated in 250-mL flasks with potato dextrose broth for 5 days at 25°C in darkness with continuous agitation. After filtration and washing in sterilized water, spore suspension was adjusted to (1 × 106 spores/mL) using a hemocytometer slide under a light microscope [15]. Each pot was inoculated with 100 mL of (1 × 106 spores/mL) [39]. Coffee plants that received treatment with sterile water acted as control. The plants were maintained in a controlled growth chamber set to 25°C, with a 12-h light period alternating with 12 h of darkness. Three replicates were used in a completely randomized design. Re-isolation was carried out from diseased seedlings and the recovered fungi were compared with the original fungal cultures used.

2.4 Fungicide Sensitivity Test

The comparative efficacy of seven fungicides (Table 3) was examined against N. falciformis utilizing the poison medium approach. The tested fungicides were used at a given dose as stated on the fungicide label. The PDA medium was autoclaved, and fungicides were added after cooling to 45°C–50°C and then dispensed into 90-mm Petri plates at a volume of 12 mL for each plate. Control PDA plates were left untreated with fungicides. After the media solidified, using a sterile cork borer, 5-mm discs from the outer edge of an actively growing fungal culture were introduced onto the plates and incubated at 24°C for 2 to 3 days. Every treatment was evaluated on three plates as replicates. Following incubation, the diameter of every fungal colony was assessed in two orthogonal directions. The growth inhibition percentage for each isolate and fungicide combination was computed utilizing the following equation:

Inhibition(%)=[(control diameter-treated diameter)/control diameter]×100%

2.5 Data Analysis

The obtained data were evaluated employing analysis of variance one-way ANOVA. The CoStat software, version 2.6 [40], was utilized to separate means through Fisher’s protected least significant difference (LSD) test at a significance level of p ≤ 0.05. The mean ± standard deviation (SD) was used to express the data.

3  Results and Discussion

3.1 Symptoms and Morphological Characterization of Neocosmospora Isolates

Overall, 12 coffee plantations were surveyed in the four screened regions. The symptoms were categorized into two main types: wilt and dry rot. Sever wilt symptoms were the most prevalent on coffee trees (Fig. 1A,B), while dry rot was less observed (Fig. 1C). Cross sections in the roots of wilted trees exhibited light to dark brown discoloration of vessels (Fig. 1D–F). Various patterns of coffee wilt symptoms attributed to different Fusarium species were reported in different literature confirming our observations [11–15]. Twenty-five fusariosis isolates were collected from plants exhibiting symptoms, and only 15 representative isolates were employed in the molecular and morphological identification. The isolates were initially classified as part of the Fusarium solani species complex. The colonies morphology on PDA were pale-cream to white-greyish, rapidly proliferating, exhibiting flat mycelium heavier near the colony’s center, and displaying a diffuse pale-yellow pigmentation that darkened with age (Fig. 2A,B). Such morphological criteria have been noted by various researchers [39,41,42]. Moreover, macroconidia conidia of N. falciformis were straight to curved, exhibiting moderate curvature and a slender form, occasionally exhibiting strong curvature (Fig. 2C). These conidia were one- to four-septate, measuring in width from 2.5 to 6.5 μm and in length from 17.5 to 35 μm. Microconidia were elongated oval and obovoid with a truncated base, predominantly non-septate, and infrequently one-septate (Fig. 2D). Spherical in shape, smooth, and thick-walled, chlamydospores were sometimes found as single entities or in pairs (Fig. 2E–G). The morphological characteristics of macro and microconidia were comparable to those recorded in earlier research [13,39,41]. Furthermore, no sporodochia was observed, which coincided with the finding of González et al. [41].

Figure 1: Sever wilt symptoms observed on the young and old coffee trees (A, B); root system of wilted trees showing rot of feeder roots and white mycelial growth of fungi (C); cross and longitudinal sections in infected roots showing internal browning with various degrees indicating the presence of N. falciformis fungi (D–F)

Figure 2: Colonies of N. falciformis incubated for one week at 25°C on PDA (A, B), macroconidia (C), microconidia (D), chlamydospores (E, F, G) Scale bars = 10 µm

3.2 Phylogenetic Analysis

The fifteen isolates obtained in this study were preliminarily classified as N. falciformis and were then chosen for the analysis of the phylogenetic. The alignment of RPB2, ITS, TEF-1α, and CaM included a total of 32 strains, which encompassed type species, representing all of the species belonging to the Neocosmospora solani clade and others from nearby clades. Fig. 3 displays the resulting ML tree. The findings revealed that isolates acquired in this study were grouped with N. falciformis CBS 475.67 with high statistical support values (a Bayes/ultrafast bootstrap% = 1.0/100%), which confirmed their taxonomic positions. The multi-locus dataset validated the formation of six sub-clades (Fig. 3). The clades 1, 4, and 5 contained two isolates, while, clades 2, 3, and 6 were made up of three isolates. This indicates a significant level of genetic variation in N. falciformis from Saudi Arabia. The reasons behind genetic diversity could be particular ecological and agricultural conditions that offer favorable conditions for the pathogen’s evolution, and the capacity of the pathogen for horizontal gene transfer [43,44]. Furthermore, the genetic diversity of these isolates may be attributed also to the fact that the genes are subject to a variety of selective pressures, which may have resulted in varying mutation rates. This may be one of the reasons for the presence of numerous clades or groups. Molecular diagnosis confidently validated the identification relying on morphological traits. Crous et al. [21] indicate that the primary genes employed for identification are TEF-1α and RPB2, which offer excellent resolution for most species across all genera. Additionally, RPB2 enhances the ability to distinguish between closely related species. This was evident from our data, employing TEF-1α, RPB2 offered high discriminatory power along with the employed markers ITS and CaM in resolving N. falciformis from other species. Such data produce well-supported phylogenetic relationships between isolates, which are in agreement with previous studies [18–21,44].

Figure 3: Maximum likelihood tree produced by phylogenetic analysis of TEF-1α, ITS, RPB2, and CaM datasets of the Neocosmospora solani clade. Numbers at the nodes signify SH-aLRT support (%)/a Bayes support/ultrafast bootstrap support (%), in that order. N. ambrosia CBS 571.94 was utilized as the outgroup. The expected nucleotide changes per site are represented by the scale bar. Sequences of N. falciformis obtained in this study are indicated with KSA letters followed by several isolates

3.3 Pathogenicity Test

All coffee seedlings inoculated with the conidial suspension of N. falciformis showed symptoms of wilt resembling those noted in the field (Fig. 4). There was no difference in the final occurrence of wilt, which was consistent across all inoculated coffee plants, with a uniform occurrence rate of 100%. Likewise, Chen et al. [45] demonstrated also that Neocosmospora silvicola caused 100% mortality in artificially inoculated detached Pinus armandii branches. Also, López-Moral et al. [46], indicated that the inoculated Prunus dulcis plants with Fusarium oxysporum under irrigation demonstrated significantly highest incidence (100%) of wilt symptoms. In this study, the initial signs manifested as either wilting or browning of leaves, leading to desiccation, loss, and finally plant death. The observed symptoms were also previously reported [15]. The isolate KSA 24-14 showed first wilt symptoms after 28 days post-inoculation, which coincided with the finding reported by Tshilenge-Djim et al. [11]. While, the other isolates (KSA 24-1, KSA 24-4, and KSA 24-6) caused first wilt symptoms after 35 days. This variation in symptom development was also noticed among Fusarium species [11,14]. There were no premature defoliated leaves observed, even after 60 days of inoculation and all leaves remained attached to infected plants. However, previous studies revealed that coffee plants treated with F. solani, F. xylarioides, F. falciforme, and F. stilboides exhibited severe defoliation [11,15]. The control plants remained symptomless (Fig. 4). The fungus was successfully recovered after the re-isolation from the wilted plants and its identification was validated by contrasting it to the original isolate, thus fulfilling Koch’s postulates. The current findings align with previously reported data suggesting that Fusarium/Neocosmospora species can induce varied degrees of wilt symptoms in inoculated coffee plants. The geographic origin and age of strains appear to be related to this variability in pathogenicity [15]. Neocosmospora falciformis is not only a plant pathogen but also an important fungus that can trigger diseases in animals and humans, mostly as an opportunistic pathogen [19]. It also encompasses aggressive plant pathogens, including the previously classified F. paranaense, a species implicated in root rot of Glycine max in Brazil that has been synonymized with N. falciformis following the phylogenetic reassessment of the genus Fusarium [19,47]. Additionally, N. falciformis, formerly known as F. falciforme, has been recognized as a pathogen affecting Phaseolus lunatus and has been linked with Fusarium wilt in Cannabis sativa, as well as causing bud and wilt rot in A. tequilana [48–50].

Figure 4: Symptoms induced by artificial inoculation of representative isolates (KSA 24-1, KSA 24-4, KSA 24-6, and KSA 24-14) of N. falciformis revealed wilt symptoms on six-month-old coffee seedlings after 35 days post-inoculation

3.4 Fungicide Sensitivity Test

Data in Table 4 reveals that the all-tested fungicides suppressed the mycelial growth of N. falciformis KSA 24-14, regardless of fungicide concentration and displaying significant variation. Only Maxim XL® exhibited the highest inhibitory action among the tested fungicides against N. falciformis KSA 24-14, reducing the mycelial growth up to 6 mm with 93.33% inhibition, followed by Moncut® which suppressed mycelial growth to 7.5 mm with inhibitory percentage of 91.67%. The high efficacy of fludioxonil toward Fusarium oxysporum was previously documented in several researches [51,52]. Nevertheless, the excessive use and improper utilization of fludioxonil has led to the emergence of resistance to this fungicide in Fusarium [53]. Our results indicated that Thiophanate-methyl was shown to be efficient also against N. falciformis, which coincided with those published in the literature [54,55]. Nevertheless, the research of Petkar et al. [56], demonstrated for the first time that F. oxysporum is resistant to thiophanate-methyl. Also, Uniform® and Rizolex-T® were moderately effective in reducing mycelial growth of N. falciformis KSA 24-14 to values of 11.4 and 15 mm, with inhibitory percentages reaching 87.36% and 83.33%, respectively.

By contrast, Captan Ultra® and Folicur® were the least efficient fungicides for inhibiting the mycelial growth of N. falciformis, with an inhibition value of 75.83% and 78.19%, respectively. This could explain why the level of pre-existing resistance to tebuconazole is high and should, thus, be regarded as intrinsic resistance. However, earlier studies reported that tebuconazole the active ingredient of Folicur® had a great inhibitory effect against F. oxysporum [57,58]. This was also reported in an earlier study that demonstrated the development of tebuconazole resistance in F. graminearum [59]. The molecular processes underlying intrinsic resistance in Fusarium remain unexplored. Nevertheless, there is limited data regarding the activity and potential resistance risks of tebuconazole and fludioxonil in N. falciformis. Thus, in this study, the sensitivity test offers initial data to assess the resistance levels in field populations, thereby guiding the strategic selection of suitable fungicides.

4  Conclusion

This study marks the initial effort to elucidate the etiology and pathogenicity of Neocosmospora species related to the wilt and deterioration of coffee trees, aiming to manage this economically significant disease and ensure the financial success of the industry of coffee in Saudi Arabia. Identifying the causal agents may facilitate the development of an appropriate management scheme predicated on their susceptibility to fungicides. We report for the first time in Saudi Arabia the potential of N. falciformis to cause decline and wilt on coffee. Our study revealed that TEF-1α and RPB2, in combination with the markers ITS and CaM, provided significant discriminatory power in distinguishing N. falciformis from other species. It is vital to monitor the emergence of fungicide resistance, as the inconsistency observed in vitro sensitivity tests underscores the importance of adapting recommendations. This approach helps prevent the continued use of ineffective fungicides. Further investigation is required to examine the differences in sensitivity to various fungicides among isolates of the same fungal species. The validation of the tested fungicides under field conditions could also be investigated in future work.

Acknowledgement: The authors express their sincere appreciation to the Deanship of Scientific Research and the Vice Presidency for Graduate Studies and Scientific Research at King Faisal University, Saudi Arabia, for their financial support of this research through grant number KFU242134.

Funding Statement: This research and APC were funded by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Saudi Arabia, for supporting this work for work through grant number KFU242134.

Author Contributions: The authors confirm contribution to the paper as follows: study conception and design: Ahmed Mahmoud Ismail, Khalid Alhudaib; data collection: Ahmed Mahmoud Ismail, Khalid Alhudaib; analysis and interpretation of results: Donato Magistà; draft manuscript preparation: Ahmed Mahmoud Ismail, Donato Magistà. All authors reviewed the results and approved the final version of the manuscript.

Availability of Data and Materials: The findings and datasets analyzed throughout this study are accessible within the published article. Additionally, they can be made available upon reasonable request.

Ethics Approval: Not applicable.

Conflicts of Interest: The authors declare no conflicts of interest to report regarding the present study.

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