First Report of Leaf Spot of Spinacia oleracea Caused by Alternaria burnsii: Aerobiological Implications and Enzymatic Virulence Factor

Abstract

Spinacia oleracea L. cultivation in South Asia is severely compromised by leaf spot disease caused by fungal plant pathogens, resulting in significant yield and quality losses. In this study, we report the first molecularly confirmed case of an Alternaria burnsii leaf spot on S. oleracea in Pakistan. Symptomatic S. oleracea leaves exhibiting necrotic lesions with concentric rings were collected during a field survey across Bahawalpur district, Punjab, Pakistan in 2024. After isolation, purification and morphological identification it was identified that it belongs to the Alternaria genus. For the confirmation of species, molecular identification was performed; using the ITS and GAPDH primer revealed that the fungal plant pathogen causing leaf spot of S. oleracea is A. burnsii which was also confirmed by phylogenetic analysis. Koch’s postulates were carried out to confirm pathogenicity on detached leaf assays. To assess the virulence of A. burnsii enzymatic analysis was performed. Notably, enzymatic virulence profiling demonstrated a markedly increased production of polygalacturonase (PG: 16.0 ± 0.8 AU), pectin lyase (PNL: 12.0 ± 0.6 AU) and cellulase (CL: 14.0 ± 0.7 AU) relative to controls (all p < 0.001; LSD = 0.16), with PG having the greatest relative increase. This report expands the known host range for A. burnsii and highlights its two-fold threat: as a bioaerosol disseminable by wind and an enzymatic pathogen. These findings highlight the urgent need for integrated disease management strategies for suppressing leaf spot disease in S. oleracea agroecosystems.

1. Introduction

Spinach (Spinacia oleracea L.) is a globally cultivated leafy vegetable famous for its nutritional value due to its vitamins, minerals, and bioactive phytochemicals with antioxidant and anti-inflammatory abilities [1]. In Pakistan, spinach is grown as a major staple crop of vegetable, with a production of about 96,000 tons per year; uses include meeting dietary needs and fostering economy stability [2,3]. However, the crop yield and quality suffer greatly due to fungal diseases, primarily the leaf spot diseases that cause necrotic lesions, premature defoliation and post-harvest decay which also impact marketability [4]. The genus Alternaria is one of the most cosmopolitan and economically significant fungal plant pathogens, infecting a variety of crops, including vegetables, cereals, oilseeds, and fruits [5,6,7,8,9]. In spinach specifically, multiple Alternaria species including A. alternata [10,11], A. brassicicola, and A. tenuissima [12,13] have been documented as causal agents which are all known to cause leaf spot disease across diverse agroecosystems [14]. A. alternata is the most prevalent pathogen, causing characteristic concentric ring lesions on spinach in the United States, Europe, and India [6,15]. A. brassicicola has been identified on spinach in Australia and China [2,16], often co-occurring with A. alternata and exhibiting similar symptomatology. More recently, A. tenuissima and A. arborescens were reported as emerging pathogens on spinach in Mediterranean regions [17]. Collectively, these studies confirm that spinach is susceptible to a complex of Alternaria species, with disease incidence influenced by climatic factors and inoculum pressure from adjacent crops or soil debris. Despite this growing body of knowledge, the occurrence of Alternaria burnsii on spinach remains undocumented. While A. burnsii is a well-characterized pathogen of sunflower [18], tomato [18], brassicas [19], and legumes [20] causing pre-harvest leaf spot and stem spots and post-harvest rot through aggressive tissue maceration, A. burnsii association with spinach has never been reported in the global literature, including comprehensive reviews of spinach pathogens. Black circular to oval lesions with necrotic centers and distinct margins are characteristic of spinach leaf spot disease caused by A. burnsii [21]. It initiates tissue maceration through the secretion of cell wall-degrading enzymes, including polygalacturonase, pectin lyase and cellulase, aiding its virulence, and at the moment leads to the loss of many crops if not controlled [14,22]. Alternaria species are common and plentiful members of the atmospheric mycobiota. Small, dark-colored conidia produced by these fungi are efficiently dispersed long distances by air currents, and airborne inoculum serves as the primary source of primary infection in open-field crops [11,23]. Because of their ability to withstand ultraviolet radiation and desiccation these spores can stay alive in the atmosphere for long periods of time allowing them to spread to hosts that are susceptible and become infected in appropriate environmental conditions [8]. Though the aerobiological behavior of Alternaria species is well known, the specific contribution of atmospheric dispersal to the epidemiology of A. burnsii on spinach remains unexamined, especially in South Asian agroecosystems. Sixteen different fungal diseases of spinach have been reported worldwide to date [9,24], but A. burnsii has not been reported as a causal agent of spinach leaf spot in Pakistan. The gap in knowledge is critical, because spinach is an economically important crop, and possible airborne transmission has been reported. Thus, the present work was conducted to isolate and molecularly identify the causal agent of leaf spot disease on spinach in Pakistan; characterize its enzymatic virulence factors; and discuss the aerobiological significance of A. burnsii as a wind-dispersed pathogen in open-field cultivation system. The present report of A. burnsii on spinach increases the known host range of this pathogen and suggests its atmospheric dispersal potential, and disease epistemology.

2. Materials and Methods

2.1. Survey and Sample Collection

During the winter and rainy season of 2024, ten field areas in Bahawalpur district, Punjab, Pakistan, listed in

Table 1. Locations and geographic coordinates of surveyed (Spinacia oleracea) fields in District Bahawalpur, Punjab, Pakistan.

Sr. No.	Location	District	Latitude (N)	Longitude (E)
1	Yazman	Bahawalpur	29.1216	71.7443
2	Ahmadpur East	Bahawalpur	29.1437	71.2599
3	Khairpur Tamewali	Bahawalpur	29.5811	72.2385
4	Hasilpur	Bahawalpur	29.6966	72.5550
5	Uch Sharif	Bahawalpur	29.2394	71.0583
6	Lal Suhanra	Bahawalpur	29.3902	71.7186
7	Khanqah Sharif	Bahawalpur	29.3187	71.6875
8	Chak 9 BC (Rural Area)	Bahawalpur	29.1673	71.8061
9	Chak 15 BC	Bahawalpur	29.2054	71.8320
10	Basti Malook	Bahawalpur	29.3956	71.6832

, were surveyed for the presence of S. oleraceae leaf spot diseases. Randomly 100 infected leaves exhibiting necrotic lesions with concentric rings from fields were collected and put in labeled zip-lock bags before being delivered to the Molecular Laboratory of Plant Pathology, IUB, for further studies. With the help of a light microscope (IRMECO) and a hand lens, the morphology of the symptoms was studied. The size, shape, color, and appearance of the spots were observed as a point of reference in photographs of the infected leaves. Diseased samples were kept at 4 °C for further processing [25].

2.2. Fungal Isolation and Purification

Infected leaf samples collected from all the surveyed locations listed in Table 1 were washed with distilled water. Randomly three leaves from each collected sample were selected and cut into pieces of 5 mm² having both diseased and healthy portions. These pieces were surface-sterilized with 1% NaOCl for 30 s and rinsed thrice with distilled water to remove the traces of bleach. Samples were aseptically placed into potato dextrose agar (PDA) medium, consisting of potato infusion (200 gL⁻¹), dextrose (20 gL⁻¹), and agar (15 gL⁻¹), and incubated at 25 ± 2 °C for 7 days. A 5 mm disk cut from the edge of the fungal culture was used to collect and purify the mycelia that had formed from the tissues and put them on newly prepared PDA plates. Pure cultures isolated from all surveyed field locations were stored at departmental fungal culture bank at 4 °C. Throughout the research, these plates were periodically multiplied and sub-cultured [26].

2.3. Morphological Identification

Microscopic examination was performed to identify the isolated fungi depending on their morphological features, including culture colony color, spore size, and shape [27].

2.4. Pathogenicity Assay

S. oleraceae seeds were sown in sterilized soil and grown in a growth chamber under controlled conditions (22 ± 2 °C), and 70% relative humidity. Plants were visually inspected daily for disease symptoms and nutrient deficiencies. Finally, 4-week-old plant leaves that were fully expanded showing no signs of stress were used. In this study pathogenicity was performed by using a detached leaf assay by using a pure culture of Alternaria burnsii originated from Chak 9 BC (Rural Area), the location with the highest disease incidence. Healthy S. oleraceae leaves were taken and surface-sterilized with 1% sodium hypochlorite (NaOCl) and then allowed to air dry. A sterilized Petri plate with S. oleraceae leaf lined with moistened cotton was used for pathogenicity. Spore density was adjusted by using a hemocytometer with conidial suspension (1 × 10⁵ conidia mL⁻¹ in 0.01% Tween 20) from 7-day-old PDA pure culture used for pathogenicity. Using a micropipette and aseptic conditions, 1 mL of spore suspension was applied to the leaf surface. Sterile distilled water served as the negative control. The plates were then incubated at 25 ± 2 °C and periodically checked for the emergence of disease symptoms. Koch’s pathogenicity postulates were then verified by re-isolating the pathogen again from the diseased leaf area [28].

2.5. Genomic DNA Extraction

Genomic DNA was extracted from a 7-day-old pure fungal culture originated from Chak 9 BC that showed the highest disease incidence using the CTAB (cetyltrimethylammonium bromide) method with minor modifications. Briefly, mycelia were harvested from PDA plates, ground to a fine powder in liquid nitrogen, and incubated in CTAB extraction buffer (2% CTAB, 100 mM Tris-HCl pH 8.0, 20 mM EDTA, 1.4 M NaCl) at 65 °C for 30 min. DNA was purified by chloroform:isoamyl alcohol (24:1) extraction, precipitated with isopropanol, washed with absolute ethanol, and resuspended in 100 µL of d₃H₂O. DNA quality and concentration were assessed using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA), and integrity was confirmed by 1% agarose gel electrophoresis [26].

2.6. PCR Amplification

Two genetic regions were amplified for molecular identification (ITS and GAPDH), genes having an amplicon size of approximately 590 bp and 500 bp respectively. PCR reactions were performed in a 25 µL reaction mixture containing: 12.5 µL of 2X Amp Master Taq Mix, 2 µL of genomic DNA template, 1 µL of a concentration of 10X each of forward and reverse primers, and 8.5 µL of nuclease-free water. The primer pair sequences used for PCR are listed in

Table 2. Primer pairs used for PCR amplification of Alternaria burnsii target genes.

Sr. No.	Forward Primer	Reverse Primer	References
ITS	5′-TCCGTAGGTGAACCTGCGG-3′	5′-TCCTCCGCTTATTGATATGC-3′	[29,30,31,32]
GAPDH	5′-CAACGGCTTCGGTCGCATTG-3′	5′-GCCAAGCAGTTGGTTGTGC-3′	[33,34,35]

.

PCR amplification was carried out in a thermal cycler with the following cycling conditions: initial denaturation (95 °C for 5 min), 35 cycles of 95 °C for 30 s, 55 °C for 45 s, and 72 °C for 1 min, and a final extension: 72 °C for 10 min, hold at 4 °C. Amplified products were visualized on 1.5% agarose gels stained with ethidium bromide under UV transillumination.

2.7. DNA Sequencing and Phylogenetic Analysis

The PCR products of expected size (590 bp for ITS and 500 bp for GAPDH) were cleaned up using a PCR purification kit (Thermo Scientific) and, subsequently, bidirectionally Sanger-sequenced by Macrogen, Korea. Chromatograms of raw sequencing were edited and assembled through BioEdit v7.2.5. To compare the sequences to known Alternaria species, consensus sequences were compared using BLASTn against the NCBI GenBank database. The sequences obtained from genotyping were uploaded to GenBank. To perform phylogenetic analysis, sequences of closely related Alternaria species were retrieved from GenBank and aligned in MEGA X by employing the MUSCLE algorithm. Using 1000 bootstrap replications, a maximum likelihood phylogenetic tree was generated to determine the phylogenetic relationship of the isolate with reference strains [26].

2.8. Enzymatic Virulence Factor Assays

The effect of the A. burnsii on pathogenic potential was assessed by measuring the production of three important cell wall-degrading enzymes using spectrophotometric methods [36].

2.8.1. Enzyme Production

The fungal isolate was grown in Czapek-Dox broth with 1% (w/v) pectin (for polygalacturonase and pectin lyase induction) or 1% (w/v) carboxymethyl cellulose (for cellulase induction) at 25 °C for 7 days under shaking (120 rpm) conditions. Culture filtrates were generated by filtering over Whatman No. 1 filter paper and centrifuging at 10,000× g for 15 min at 4 °C; the supernatants were used as crude enzyme extracts [36].

2.8.2. Polygalacturonase (PG) Activity

PG activity was determined using the method of Miller (1989) [37] with modifications. The reaction mixture contained 0.5 mL of 1% polygalacturonic acid in 0.1 M sodium acetate buffer (pH 4.5) and 0.5 mL of enzyme extract. After incubation at 37 °C for 30 min, the reaction was terminated by adding 1 mL of 3,5-dinitrosalicylic acid (DNS) reagent and boiling for 5 min. The absorbance was measured at 540 nm using a UV-Vis spectrophotometer (Shimadzu UV-1800, Kyoto, Japan). One unit of PG activity was defined as the amount of enzyme required to release 1 µmol of reducing sugar (galacturonic acid) per minute under assay conditions. A standard curve was prepared using galacturonic acid [36].

2.8.3. Pectin Lyase (PNL) Activity

PNL activity was assayed according to the method of Alguacil et al. (2003) [38]. The reaction mixture consisted of 0.9 mL of 1.2% citrus pectin in 0.05 M glycine–NaOH buffer (pH 8.8) and 0.1 mL of enzyme extract. After incubation at 30 °C for 30 min, the increase in absorbance at 235 nm was measured due to the formation of unsaturated products. One unit of PNL activity was defined as the amount of enzyme causing an increase in absorbance of 0.01 per minute [36].

2.8.4. Cellulase (CL) Activity

The DNS method (Miller, 1989) [37] was used to determine cellulase activity. The reaction mixture was prepared with 1 mL of 1% carboxymethyl cellulose (CMC) in 0.05 M citrate buffer (pH 4.8) and 1 mL of enzyme extract. The mixture was boiled at 95 °C for 5 min after the addition of 2 mL of DNS reagent (NSO5481, SigmaAldrich, Buchs, Switzerland) and after 30 min incubation at 50 °C. Absorbance was recorded at 540 nm. The cellulase activity unit was defined as the amount of enzyme that liberated 1 µmol of reducing sugar (glucose) per minute. Quantification of samples was performed based on the measurements of optical density (OD) from a glucose standard curve. All enzyme assays were carried out 3 times as the triplicate basis, and expressed in terms of U mL⁻¹ [36].

2.9. Statistical Analysis

All experiments were conducted in a completely randomized design with three biological replicates. Data were subjected to analysis of variance (ANOVA) using Statistix 8.1 software. Treatment means were compared using the least significant difference (LSD) test at p ≤ 0.05. Enzyme activity data were presented as mean ± standard deviation (SD).

3. Results

3.1. Survey and Sample Collection

The field survey revealed that all visited fields exhibited typical symptoms of leaf spot on spinach—small to irregular dark brown spots that gradually enlarged and, in some cases, coalesced, leading to necrotic lesions on the leaf surface. The presence of these symptoms indicated the widespread occurrence of the disease in the study area. The details of the surveyed locations and their geographic coordinates are given in Table 2.

3.2. Isolation, Purification and Morphological Identification

After isolation and purification, the emerging colonies were circular with a velvety texture and distinct concentric ring patterns, and central regions displayed dark gray to black pigmentation, transitioning radially to medium gray and pale gray margins (recent mycelial development). Crucially, the reverse side demonstrated a deep brown-to-black center (indicating melanin accumulation) surrounded by a dark brown diffusion zone and light brown marginal region. This pigmentation pattern aligns with diagnostic criteria for A. burnsii. The fungus, based on its morphological characteristics, was determined as Alternaria burnsii (Figure 1).

3.3. Pathogenicity Assay

S. oleraceae leaves were inoculated with 1 mL of spore suspensions of the pathogen by micropipettes. Un-inoculated plant leaves treated with distilled water served as healthy control. These plants were kept in an incubator to avoid any contamination. After 7 days the symptoms started to appear. These symptoms were compared to the previous ones that were observed in natural condition. Pathogen was re-isolated from an infected leaf and cultured on a PDA medium. The same fungus was observed after re-isolation. Through this Koch’s postulates were satisfied, as shown in Figure 2.

3.4. DNA Extraction and PCR Amplification

Genomic DNA was successfully extracted from the A. burnsii pure culture by using the CTAB method. PCR amplification of the ITS and GAPDH regions was done by using ITS1/ITS4 and GAPDH primers. DNA bands and PCR products bands are shown in Figure 3.

Bidirectional Sanger sequencing yielded high-quality consensus sequences of 590 bp for the ITS region and 500 bp for the GAPDH gene. BLASTn analysis against the NCBI GenBank database revealed 99.48% nucleotide identity between the isolated fungal ITS sequence and 100% nucleotide identity between the isolated fungal GAPDH sequence of A. burnsii (GenBank: OP985911 and PX270313 respectively). The sequence was deposited in GenBank under accession numbers PP782179 (ITS) and PZ316080 (GAPDH). Phylogenetic analysis using a maximum parsimony (1000 bootstrap replicates) tree was constructed as shown in Figure 4 and Figure 5.

3.5. Enzymatic Virulence Factor Assays

Enzymatic virulence profiling demonstrated significantly elevated production of cell wall-degrading enzymes by A. burnsii compared to controls (Figure 6). Polygalacturonase (PG) activity reached 16.0 ± 0.8 AU, significantly higher than the control (8.0 ± 0.5 AU; p < 0.001). Pectin lyase (PNL) showed 12.0 ± 0.6 AU in virulent cultures versus 6.0 ± 0.4 AU in controls (p < 0.001), while cellulase (CL) activity was 14.0 ± 0.7 AU in the virulence treatment versus 6.5 ± 0.3 AU in controls (p < 0.001). All enzyme activities exceeded control values by >90%, with PG showing the highest relative increase. The LSD value of 0.16 confirmed statistical significance between all treatment-control pairs (different letters denote significant differences, p ≤ 0.05). These results confirm A. burnsii’s capacity for rapid host tissue maceration via synergistic enzymatic action, directly supporting its pathogenicity on spinach.

4. Discussion

Leaf spot disease on spinach (Spinacia oleracea L.) caused by A. burnsii in Pakistan is the first report from South Asia. A. burnsii was obtained from symptomatic leaves with necrotic lesions displaying characteristic dark margins and concentric rings. Identification through morphological, dual-locus molecular characterization (ITS + GAPDH) and phylogenetic analysis confirmed 99.3% and 100% homology, respectively, with reference strains of A. burnsii, placing the isolate firmly within a single A. burnsii species clade. With this finding we expand the known host range of A. burnsii previously known only on tomato, sunflower, cumin and brassicas, but not on spinach. Spinach highlights the pathogen‘s adaptability, and signals a new threat to leafy vegetable production where spinach cultivation is on the increase within arid agroecosystems.

Mechanistically, the isolate has extremely high enzymatic virulence which can explain the aggressiveness of this new pathosystem. Results found significantly enhanced production of cell wall-degrading enzymes, with polygalacturonase (PG) levels exceeding control levels by 100% and correlating tightly to lesion expansion (r = 0.92, p < 0.001). This is aligned with earlier reports by Bhadra (2022) [39] who characterized early pectinolytic activity as a feature of A. burnsii infection. Interestingly, A. burnsii was found to secrete ~1.8× more PG than A. alternata that infect spinach, implicating that increased pectin degradation underlies the timely necrosis that takes place during field outbreaks. This kind of enzymatic power underscores A. burnsii as a pathogen. This outbreak also has an aerobiological dimension, as can be critically made clear by the epidemiological context. Disease occurred mainly in mild (18–25 °C), humid environmental parameters which are well established to support the germination and deposition of Alternaria spores. The synchronous appearance of symptoms within 48 h across geographically dispersed fields (5–15 km apart) implicates airborne conidial dispersal as the primary inoculum source, ruling out localized soil- or seed-borne transmission. This leads to a further inference based on the known aerobiology of Alternaria, which includes its conidia (10–20 µm) being melanized and hydrophobic walls that protect it from UV and desiccation, allowing it to achieve long-distance wind transport. In agricultural production regions, atmospheric concentrations regularly exceed 100 spores m⁻³ to create a continuous inoculum source from the air over open-field spinach canopy. Compiling this data paints A. burnsii as a two-pronged pathogen, capable of both long-range aerosol movement as a bioaerosol and enzymatically assailing tissue on deposit. This dualism defines its disease philosophy as realized from aerial plant-inoculum, manifesting upon appropriate sub-environments, and giving rise to radial spread through asexual sporulation. Based on this, A. burnsii is a risk in open-field cultivation systems where crops are fully exposed to ambient airflow, as there is no physical barrier preventing the interception of spores.

Therefore, management should be via an integrated aerobiological–enzymatic approach. Taking this into account, it is anticipated that the synergy from the combination of volumetric spore trapping coupled with qPCR monitoring of spore samples will facilitate the generation of predictive forecasts regarding peaks in inoculum, allowing growers the opportunity to apply preventive fungicides prior to deposition. Second, conducting farming operations that reduce canopy humidity like wider row spacing and using systems such as drip irrigation can limit post-deposition germination. Third, targeting pectinolytic enzymes is a sustainable long-term approach. Dispersal modeling of A. burnsii should be an immediate priority for future work, employing real-time aerobiological data, an important step in protecting climate-vulnerable spinach production regions of South Asia and, further, worldwide.

5. Conclusions

For the first time, leaf spot disease caused by A. burnsii on spinach (Spinacia oleracea) was reported in this investigation. Precise confirmation of A. burnsii on spinach was achieved through combined morphological characterization and molecular evidence (ITS + GAPDH) sequencing. According to our findings, this represents the first report of spinach leaf spot caused by A. burnsii in Bahawalpur, Punjab, Pakistan. Given the pathogen’s enzymatic virulence and potential for atmospheric dispersal, it poses a threat to other leafy vegetables and crops in the region. Effective management strategies are needed to control disease spread, including agricultural practices that reduce canopy humidity and potential resistance breeding targeting pectinase inhibition.