Alternaria Species Research Articles

Baseline Sensitivity and Toxicity Mechanisms of Prochloraz to Alternaria alternata Strains Associated with Maize Leaf Blight in Heilongjiang Province in China.

Alternaria species are fungal pathogens that can infect maize, causing leaf blight disease and significant economic losses. This study aimed to determine the baseline sensitivity to prochloraz of A. alternata isolates obtained from diseased maize leaves collected from Heilongjiang Province by assessing the half-maximal effective concentration (EC50) values. The EC50 values of prochloraz ranged from 0.0550 to 2.3258 μg/ml, with an average of 0.9995 ± 0.5192 μg/ml. At EC50 (1.2495 μg/ml) and 2EC50 (2.4990 μg/ml), prochloraz increased the number of mycelial offshoots, disrupted the cell membrane integrity of conidia and mycelia, and resulted in a reduced ergosterol content in the mycelia. Prochloraz significantly affected the mycelial cell membrane permeability and increased the malondialdehyde content and superoxide dismutase activity. No cross-resistance was detected between prochloraz and other fungicides. These data demonstrate that prochloraz is a promising fungicide for managing maize leaf blight caused by A. alternata and provide novel insights into understanding the mechanism of prochloraz toxicity against A. alternata isolates.

First report of Alternaria leaf spot caused by Alternaria brassicae on broccoli in North Carolina, United States.

In September 2023, broccoli (Brassica oleracea var. italica) 'Sweet Bunch' plants on an organic farm in Buncombe County, North Carolina (NC), displayed symptoms of Alternaria leaf spot. Disease affected 10 to 20% of leaf area on all (approximately 30) plants. Lesions were dark brown with chlorotic halos, irregular in shape, and ≤3 cm in diameter. Leaf samples were incubated in a humidity chamber for 24 h, and the pathogen was putatively identified as Alternaria brassicae based on conidial morphology (Rimmer et al. 2007; Supplemental Fig. 1). Leaf pieces (2 x 2 mm) spanning lesion edges were surface-disinfested (1% sodium hypochlorite solution) for 1 min, rinsed in sterile distilled water, embedded in potato dextrose agar, and incubated at 22°C with a 12-h photoperiod. A pure culture was obtained by transferring a hyphal tip onto 20% V8 juice agar and incubated as above. After 10 d, colonies consisted of concentric zones of white to brown mycelia with white, aerial mycelia. Within 7 d, conidia measuring 80 to 150 µm long with 0 to 3 longitudinal septa, 6 to 15 transverse septa, and a long beak were observed, and the isolate was putatively identified as A. brassicae (Rimmer et al. 2007). To confirm identification, DNA was extracted from mycelia and subjected to PCR using primers to amplify a portion of the internal transcribed spacer of rDNA (ITS: ITS1/ITS4) (White et al. 1990), the transcription elongation factor-1 (tef1: EF1-728F/EF1-986R) (Carbone and Kohn 1999), and the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH: gpd1/gpd2) (Berbee et al. 1999). Amplicons were sequenced at Eton Bioscience (Durham, NC) and subjected to BLAST analysis in NCBI GenBank. Sequences matched (99.5 to 100% identity) A. brassicae CBS 116528 accessions KC584185 (ITS), KC584641 (tef1), and KC584102 (GAPDH) (Woudenberg et al. 2013). All sequences were deposited in NCBI GenBank (accessions PP584506, PP584507, PP584508). To confirm pathogenicity on broccoli and kale (B. oleracea var. sabellica), 5-week-old plants of broccoli 'Sweet Bunch', 'Eastern Crown', and 'Green Magic' and kale 'Winterbor', 'Darkibor', and 'Oldenbor' grown in 10-cm-diameter pots were separated by crop type and arranged in four randomized complete blocks by cultivar. Each block contained single-plant replicates of each cultivar that were either inoculated or non-inoculated. Conidia of A. brassicae obtained from 10-d-old cultures (3.5 x 103 conidia/ml) were sprayed onto each inoculated plant until run-off. Non-inoculated plants were sprayed with distilled water. Plants were incubated in a humidity chamber alternated with ambient greenhouse conditions every 48 h for 10 d, with average greenhouse temperatures ranging from 15 to 28°C. The experiment was conducted twice. Dark brown to gray lesions measuring 1 to 3 mm in diameter were observed on leaves of all inoculated plants within 5 d of inoculation (Supplemental Fig. 2), and non-inoculated plants remained healthy. On broccoli plants, lesions expanded in concentric rings, and sporulation of the pathogen was observed on leaf tissue and petioles 10 d after inoculation. Lesions remained small (< 3 mm) on kale plants, and sporulation was not observed. Alternaria brassicae was re-isolated from leaf tissue of all inoculated cultivars using methods described above. Pathogen identity was confirmed via colony and conidial morphology. The larger impact of A. brassicae on brassicas is unknown, but finding this less common Alternaria species in the eastern United States could have implications for host resistance development and fungicide efficacy (Nieto-Lopez et al. 2023).

Response of Alternaria and Fusarium Species to Low Precipitation in a Drought-Tolerant Plant in Morocco

The plant mycobiome plays a crucial role in the host life cycle, influencing both healthy and diseased states, and is essential for plant tolerance to drought. In this study, we used ITS metabarcoding to investigate the fungal community of the drought-resistant plant Malva sylvestris L. in Morocco along a gradient of precipitation, encompassing subhumid and semi-arid environments. We sampled three biotopes: rhizosphere, bulk soil, and root endosphere. Our findings revealed an absence of beta-diversity differences between bulk soil and rhizosphere, indicating that the plant does not selectively influence its rhizosphere mycobiome. Additionally, ASVs belonging to the genus Alternaria represented up to 30% of reads in the plant’s roots and correlated with drought (p = 0.006), indicating a potential role for this fungal genus in mitigating drought, possibly as part of the dark septate endophyte group. Root staining and microscopic observation revealed extensive colonization by fungal hyphae and microsclerotia-like structures. Furthermore, ASVs identified as Fusarium equiseti were also correlated with low precipitation and recognized as a hub taxon in the roots. However, it remains uncertain whether this species is pathogenic or beneficial to the plant. These insights contribute to our understanding of the plant mycobiome’s role in drought tolerance and highlight the importance of specific fungal taxa in supporting plant health under varying environmental conditions. Future research should focus on characterizing these taxa’s functional roles and their interactions with the host plant to further elucidate their contributions to drought resistance.

Biodiversity of Aspergillus Species and Their Mycotoxin Production Potential in Dry Meat.

This study aimed to examine fungi diversity in dried beef meat sold in Ekiti State, characterize the isolated fungi, and determine the aflatoxin-producing ability of the Aspergillus fungi in the samples. Dried beef meat was collected from different markets in Ekiti State and screened for the presence of filamentous fungi using molecular methods. Samples were cultured aseptically on potato dextrose agar (PDA) for fungi isolation, and molecular identification was performed using DNA extraction, Polymerase chain Reaction (PCR), ITS-1/ITS-4 primer pair, and nucleotide sequencing. The results obtained indicated a range of filamentous fungi genera including Aspergillus, Rhizopus, Penicillium, Fusarium, Cladosporium, Alternaria, and other fungi species contaminating the dried meat at (43%), (42%), (3%), (2%), (2%), (1%), and (7%), respectively. High incidences were recorded for Aspergillus flavus, Aspergillus niger, and Aspergillus fumigatus in most of the screened samples. Aspergillus flavus accounted for (24.7%) of all the Aspergillus species isolated with the presence of the gene needed for aflatoxin production. The occurrences of these filamentous fungal species pose a cause for concern, as most of these fungal species are known producers of certain toxic substances. Maximum likelihood phylogenetic analysis showed a high similarity index score, which indicated a good relationship between isolated Aspergillus Species and the closely related strains from GenBank, isolated from different sources and countries. The implication of this study is that consumer health may be at risk through exposure to contaminated dried meat.

Effect of bloom sprays on Alternaria rot in California mandarins and fungicide efficacy against Colletotrichum.

This study aimed to provide new insights into the management of Alternaria rot and Colletotrichum dieback in mandarins. Field trials were conducted to evaluate the impact of bloom sprays on latent infections, disease progression, and the influence of bloom inoculations on disease development. Detached citrus leaves were used to assess the efficacy of commercial fungicides in reducing infections by Colletotrichum spp. C. karstii was more sensitive than C. gloeosporioides, as evidenced by the overall probabilities of infection. Preventative treatments containing QoI fungicides, such as Pristine, Quadris Top, and Luna Sensation, consistently reduced infections. Curative treatments with Pristine and Luna Sensation also demonstrated efficacy. Bloom inoculations with Colletotrichum species did not induce visible symptoms on flowers and twigs, nor did they lead to dieback until harvest, suggesting that bloom infections may not play a significant role in the disease cycle. For Alternaria, the frequency of latent infections in flowers increased significantly during the bloom period. Shoots inoculated with Alternaria species during bloom reduced the number of fruits per shoot at harvest. Fungicide treatments during bloom exhibited a dual effect, reducing Alternaria infections in fruitlets and influencing disease progression, ultimately reducing preharvest fruit drop. This study provides valuable insights into the role of flower infections in the development of Alternaria rot and Colletotrichum dieback in California mandarins. Furthermore, it offers insights into the efficacy of commercial fungicides in these pathosystems.

First report of Diaporthe humulicola causing halo blight of hop (Humulus lupulus L.) in Prince Edward Island, Canada

This study documents the first report of halo blight of hop (Humulus lupulus L.), caused by the fungal pathogen Diaporthe humulicola, in Prince Edward Island (PE), Canada. This pathogen has been previously reported in Michigan, New York, Connecticut and Quebec. Disease symptoms were reported in hopyards in Harrington and Mount Albion, PE. Symptoms reported include both foliar lesions and severe necrosis of hop cones. Through a metagenomic approach using Nanopore sequencing of the full Internal Transcribed Spacer (ITS) region products, multiple fungal species were identified in infected hop cones. Many plant pathogens were present in the hop cones including species of Botrytis, Alternaria, Colletotrichum and Fusarium. Additionally, metagenomic sequencing identified reads that most closely matched D. humulicola. Symptomatic leaf tissue sections were cultured on amended PDA to isolate the pathogen, then colony PCR was conducted and ITS regions were sequenced, confirming the identity of D. humulicola. Pathogenicity was validated using Koch’s postulates on hop leaf disks, with lesion development occurring 7 days post-inoculation. Considering the humid conditions characteristic of eastern North American hop-growing zones, halo blight’s emergence in PE emphasizes the need for comprehensive monitoring and improved best management strategies to protect the PE hop industry.

Assessing Broccoli Cultivar Susceptibility to Alternaria brassicicola in Georgia

Alternaria leaf blight and head rot (causal agent: Alternaria brassicicola and other Alternaria species) in broccoli (Brassica oleracea L. var. italica) can lead to decreased yields, reduced head quality, and postharvest losses. While cultural practices and fungicides can decrease disease severity, further work is needed to determine resistance or susceptibility in commercial cultivars. This study evaluated susceptibility of 26 cultivars against A. brassicicola in Fall 2021 and 2022 at two locations in Georgia (Tifton and Watkinsville). Commonly used cultivars in the Eastern U.S. were selected and grown in a randomized complete block design. Approximately 20 days after transplant, plants were inoculated with a conidial suspension of A. brassicicola and monitored for disease development. Foliar disease severity, foliar area under disease progression curve (AUDPC), head disease severity, and yield were determined for each cultivar at each location. Significant differences existed among cultivars (year × location) for foliar AUPDC and head disease severity. Foliar AUPDC ranged from 299 to 1818 for both years and locations and head disease severity ranged from 1 to 81% across both years and locations. In this study, cultivars, ‘Eastern Crown’, ‘Emerald Jewel’, ‘Lieutenant’ and ‘Abrams’, had greater foliar and head disease severity and low marketable yields in both locations and years. The cultivars ‘Vallejo’, ‘Emerald Pride, ‘Burney’, ‘Belstar’ ‘Iron Man’, and ‘Marathon’ exhibited low disease severity across both locations and years and maintained greater marketable yields. Proper selection of commercially-available cultivars could help growers maintain yields in areas with high A. brassicicola pressure.

Etiology of Foliar Blight of Indian Paintbrush (Castilleja tenuiflora) in Mexico.

Castilleja tenuiflora is a native perennial plant used in traditional Mexican medicine. In June 2022, leaf blight symptoms were observed in a wild population of C. tenuiflora plants. Disease incidence was 80% and disease intensity reached up to 5% of the leaf area. Currently, there are no reports of pathogens causing leaf blight in this plant; therefore, this work aimed to identify the fungi responsible for the disease. The fungi recovered from the diseased tissue were characterized by means of pathogenicity tests and cultural, morphological, and molecular characterization. The information obtained revealed that Alternaria alternata and Alternaria gossypina are the pathogens responsible for the disease. This is the first report implicating species of Alternaria in causing leaf blight of C. tenuiflora in Mexico, as well as the first report of Alternaria gossypina also in Mexico. These pathogens may threaten the in situ conservation of native C. tenuiflora populations and limit their in vitro propagation. Future research lines should focus on determining the effect of these pathogens on metabolite production.

The occurrence of tenuazonic acid in food products: a systematic review

Tenuazonic acid (TeA) is a secondary toxic metabolite produced by fungi species in the genus Alternaria. It is frequently reported as an ‘emerging mycotoxin’, a group of mycotoxins which have been neither routinely determined nor legislatively regulated. This study aimed at systematically review the occurrence data of TeA in food products from 2011 to date. TeA was detected in numerous foodstuffs from several countries, especially in tomato products, herbs, spices, and cereals. Further studies are needed to understand the occurrence patterns of TeA in different foodstuffs, to orientate preventive measures to avoid Alternaria species and TeA in food production environments.

Rapid Detection of Viral, Bacterial, Fungal, and Oomycete Pathogens on Tomatoes with Microneedles, LAMP on a Microfluidic Chip, and Smartphone Device.

Rapid detection of plant diseases before they escalate can improve disease control. Our team has developed rapid nucleic acid extraction methods with microneedles and combined these with loop-mediated amplification (LAMP) assays for pathogen detection in the field. In this work, we developed LAMP assays for early blight (Alternaria linariae, A. alternata, and A. solani) and bacterial spot of tomato (Xanthomonas perforans) and validated these LAMP assays and two previously developed LAMP assays for tomato spotted wilt virus and late blight. Tomato plants were inoculated, and disease severity was measured. Extractions were performed using microneedles, and LAMP assays were run in tubes (with hydroxynaphthol blue) on a heat block or on a newly designed microfluidic slide chip on a heat block or a slide heater. Fluorescence on the microfluidic chip slides was visualized using EvaGreen and photographed on a smartphone. Plants inoculated with X. perforans or tomato spotted wilt virus tested positive prior to visible disease symptoms, whereas Phytophthora infestans and A. linariae were detected at the time of visual disease symptoms. LAMP assays were more sensitive than PCR, and the limit of detection was 1 pg of DNA for both A. linariae and X. perforans. The LAMP assay designed for early blight detected all three species of Alternaria that infect tomato and is thus an Alternaria spp. assay. This study demonstrates the utility of rapid microneedle extraction followed by LAMP on a microfluidic chip for rapid diagnosis of four important tomato pathogens.

First Report of Alternaria alternata causing leaf spot on Chinese quince (Chaenomeles sinensis (Thouin) Koehne) in Korea.

The Chinese quince (Chaenomeles sinensis (Thouin) Koehne), belongs to the Rosaceae family, is widely distributed throughout Asia, including Republic of Korea. It is used as a traditional treatment for asthma, common cold, and dry pharynx. Numerous recent pharmacological studies on antiinfluenza, antioxidant, and antidiabetic properties have confirmed the medicinal properties of the Chinese quince fruit (Chun et al., 2012). In March 2022, leaf spots on Chinese quince, resulting in defoliation, were observed in Andong, Gyeongsangbuk Province, Korea (Fig. 1A). The disease symptoms are dark brown spots on leaves. Later, the chlorophyll is lost, causing the entire leaf to become wilted and fell off (Fig. 1B). To identify the pathogen, symptomatic leaves were brought to the laboratory, cut into small pieces, and surface-disinfected in 70% ethanol for 15 s and rinsed with sterile distilled water (SDW). The specimens were then treated with 1% NaOCl for 15 s, followed by rinsing with SDW. Thus, surface-disinfected tissues were placed onto potato dextrose agar (PDA) plates and incubated at 25°C for 7 d. A total of four isolates were obtained from the infected leaves. The colonies were transferred onto freshly prepared PDA plates by the single spore method for further purification. GYUN-10746 isolate was selected as the representative strain among the four isolates and deposited in the Korean Agricultural Culture Collection (KACC 410367). They initially produced white mycelia, which turned dark brown or pale brown at the center and beige at the periphery after 7 d (Fig. 1C and D). Conidiophores were pyriform, sometimes ovoid, or ellipsoidal and brown, measuring 30.8 ± 0.49 × 12.9 ± 0.26 µm (length × width) (n=100) (Fig. 1E). The morphological characteristics were consistent with those of Alternaria alternata (Woudenberg et al. 2015). For molecular identification, DNA was amplified using the following primers: ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone et al. 1999), Gpd-R/Gpd-F (Berbee et al. 1999), Alt a1-F/Alt a1-R (Hong et al. 2005) and rpb2F/rpb2R (Liu et al. 1999) by PCR. DNA sequences from all 4 isolates (GYUN-10746, GYUN-11193, GYUN-11194 and GYUN-11195) were identical. The ITS (OP594615), TEF1-α (OR327062), GAPDH (OR372157), Alt a 1 (OR327061), and RPB2 (OR352741) sequences from the representative isolate GYUN-10746 were 100% identical to those of previously identified A. alternate isolates. A phylogenetic tree was constructed using sequences of ITS, TEF1-α, GAPDH, Alt a l, and RPB2 to illustrate their relationship with A. alternata and related Alternaria species (Fig. 2). For the pathogenicity test, healthy Chinese quince branch containing leaves were inoculated with 7-day-old mycelial plugs of A. alternata, while leaves on a branch inoculated with PDA plugs alone served as a control group. Thus inoculated branches were incubated at 25°C for 7 d. Disease symptoms were developed on leaves of the branches inoculated with mycelial plugs of the fungal pathogen (Fig. 1F), while no symptoms developed on control group. The resulting leaf spots resembled those on the original infected plants. To confirm Koch's postulates, the pathogen was re-isolated from inoculated leaves with identical morphological and molecular characteristics. To the best of our knowledge, this is the first report of leaf spot caused by A. alternata in C. sinensis in Korea. The identification of the pathogen may provide pertinent information for the development of disease controlling strategies.

Morphological and phylogenetic analyses reveal two new Alternaria species (Pleosporales, Pleosporaceae) in Alternaria section from Cucurbitaceae plants in China.

Alternaria species are commonly found as saprophytes, endophytes and plant pathogens. During a survey of small-spored Alternaria in China, two new species were discovered from Cucurbitaceae plants collected in Hubei and Sichuan provinces. This study identified two new species of Alternaria using seven genes (ITS, GAPDH, TEF1, RPB2, Alt a 1, EndoPG, and OPA10-2) for phylogenetic analyses and morphological characteristics. The two new species A.jingzhouensis and A.momordicae were described and illustrated. Alternariajingzhouensis sp. nov., associated with Citrulluslanatus, is characterized by producing muriform, ellipsoidal, flask-shaped, rostrate, and beaked conidia. It differs from A.koreana, A.ovoidea, and A.baoshanensis by bearing conidia in a simple conidiogenous locus with occasionally longer beaks in a chain, and from A.momordicae sp. nov. by having shorter beaks. Alternariamomordicae sp. nov. from Momordicacharantia was distinct from A.koreana, A.ovoidea, and A.baoshanensis by producing muriform, long ellipsoid or ovoid to obclavate, sometimes inverted club-shaped conidia on a single conidiogenous locus with a wider body and longer beak in a chain, and distinct from A.jingzhouensis sp. nov. by a longer beak conidia. These two species were clearly distinguished from other species in the section Alternaria based on DNA based phylogeny and morphological characteristics. The morphological features were discussed and compared to relevant species in the present paper.

Tenuazonic acid-induced mycotoxicosis in an immunosuppressed mouse model and its prophylaxis with cinnamaldehyde

Patients with impaired immune systems are particularly vulnerable to infections. With the increasing number of immunocompromised patients, it becomes necessary to design studies that evaluate the effects of toxic contaminants that are a part of our daily lives. Simultaneously, the management of these toxic components also becomes essential. Therefore, the present study evaluated the possible protective role of cinnamaldehyde (Cin) against tenuazonic acid-induced mycotoxicosis in the immunosuppressed murine model. Tenuazonic acid (TeA), a toxin usually produced by Alternaria species, is a common contaminant in tomato and tomato-based products. Evaluating the potential toxicity of a hazardous chemical necessitates the use of in vitro, in vivo, and in silico methods. Here, the immunomodulatory effect of TeA was assessed in vitro using mouse splenocytes. In silico docking was carried out for the tumour markers of eight organs and TeA. The haematological, histopathological, and biochemical aspects were analysed in vivo. The sub-chronic intoxication of mice with TeA showed elevated malondialdehyde, reduced catalase, and superoxide dismutase production, along with abnormal levels of aspartate aminotransferase and alanine transaminase. The treatment with Cin prevented TeA-induced alterations of antioxidant defense enzyme activities and significantly forbade TeA-induced organ damage, showing therapeutic effects and toxicity reduction in TeA-induced mycotoxicosis.

Microbial Assessment of Spoilt Tomatoes (Solanum lycopersicum Linn) Being Sold in Some Markets in Ondo City, Nigeria

Tomatoes (Solanum lycopersicum Linn) are one of the most consumed fruits in Nigeria and the world at large but they are highly susceptible to microbial spoilage. Consumption of these spoilt tomatoes could increase the risk of food poisoning and also cause serious health issues which are of public health concern. This study was therefore designed to investigate the bacteria and fungi associated with spoilt tomatoes being sold in some selected markets in Ondo city. Different media (Nutrient, MacConkey, Mannitol Salt, Salmonella –Shigella, Eosin Methylene Blue, Sorbitol MacConkey and Potato Dextrose agar) were used for the determination of the total viable bacterial and fungal counts from the samples collected from Odoshida, Adeyemi and Iyalaje markets in Ondo using the pour plate technique. The isolates obtained after subculturing were characterized based on their cultural, morphological and biochemical characteristics. The total bacterial count ranged from 1.25 ×108 to 8.06 ×108cfu/mL while the total fungal count ranged from 2.75 ×105 to 5.65 ×106 cfu/mL. The identified bacteria obtained were Staphylococcus species, Proteus species, Salmonella species, Pseudomonas species, Escherichia coli, Klebsiella species, Shigella species, Enterobacter species and Bacillus species, while the fungi were Alternaria species, Penicillium species, Aspergillus species, and Rhizopus species. This study has shown that all the isolates obtained from the selected samples are of public health concerns because of their pathogenic and toxigenic nature; an indication that spoilt tomatoes ordinarily should not be consumed or consumed with utmost caution.

Identification of Alternaria species associated with purple blotch disease of onions in Southern Punjab of Pakistan and evaluation of plant-based fungicides for disease control

Identification of Alternaria species associated with purple blotch disease of onions in Southern Punjab of Pakistan and evaluation of plant-based fungicides for disease control

Aggressive Alternaria brassicicola with Reduced Fungicide Sensitivity Can Be Associated with Naturally Infested Broccoli Seeds.

Alternaria brassicicola is a part of the Alternaria complex that causes leaf blight and head rot (ABHR) in brassica crops. Infested broccoli seeds can play an important role in introducing A. brassicicola in transplant houses and production fields. However, characterization of natural seed infestation and seed-to-seedling transmission of A. brassicicola in broccoli is yet to be demonstrated. In this research, we characterized Alternaria spp. isolates from commercial broccoli seedlots for their species identity, pathogenicity, and aggressiveness on broccoli and their sensitivity to a quinone-outside inhibitor (QoI) fungicide (azoxystrobin). Two hundred commercial seedlots from two broccoli cultivars, Cultivar 1 (EC; n = 100 seedlots) and Cultivar 2 (ED; n = 100 seedlots) were, evaluated for the presence of A. brassicicola under in vitro conditions using a seedling grow-out assay. Alternaria spp. was detected in 31 and 28% of the commercial seedlots of Cultivar 1 and Cultivar 2, respectively. The seed-to-seedling transmission (%) varied considerably within each positive-infested seedlot, which ranged from 1.3 to 17.3%. Subsequent molecular identification of single-spore cultures (n = 138) was made by sequencing four housekeeping genes: actin, the major allergen (Alta1), plasma membrane ATPase, and glyceraldehyde-3-phosphate dehydrogenase (GPD), and the sequences were concatenated and compared for the phylogenetic distance with diverse Alternaria species. Ninety-six percent (n = 133) of the isolates formed a cluster with a known A. brassicicola based on a multigene phylogeny, which were later confirmed as A. brassicicola using a species-specific PCR assay. One hundred percent of the A. brassicicola seed isolates (n = 133) were either highly or moderately aggressive on broccoli (cultivar Emerald Crown) based on a detached leaf assay. Sensitivity of representative A. brassicicola isolates (n = 58) to azoxystrobin was evaluated using a spore germination assay, and the EC50 values (effective fungicide concentration [ppm] at which germination of conidia of isolates were reduced by 50% compared to control) for each isolate was determined. A. brassicicola isolates from naturally infested commercial broccoli seeds were sensitive to azoxystrobin with considerably low EC50 values in the range of <0.0001 to 0.33 ppm; however, there were a few isolates (14%) that showed 100-fold reduced sensitivity from the most sensitive isolate (EC50 = 0.0001 ppm). Our results confirm that commercial broccoli seedlots can be naturally contaminated with pathogenic and aggressive A. brassicicola. We also provide evidence for the potential presence of A. brassicicola isolates with reduced azoxystrobin-sensitivity in naturally infested commercial broccoli seedlots, which has never been reported before. Together, these findings may have implications in considerations for seed-health testing, seed treatments, and greenhouse scouting to limit introduction of infested seedlots in commercial broccoli fields.

Inhibitory effect of plant essential oils on controlling Alternaria species

The use of natural products in the control of fungal diseases in plants is considered an alternative to synthetic fungicides due to their less negative effects on the environment. In this study, in vitro inhibitory effect of plant essential oils (PEOs) of black cumin, cumin, chamomile, cedarwood, and ginger were investigated for controlling two species of Alternaria, including Alternari solani and Alternaria alternata on tomato and cabbage under in vitro conditions, respectively. Aiming to evaluate the mycelial growth of the pathogen, mycelial discs were placed in Petri plates with 0, 500, 1000, 1500, 2000, and 2500 μL/L of PEOs. The experiment was carried out in a randomized plot design with three replications. Chemical analysis of PEO components were determined by Gas Chromatography and Mass Spectrometry methods. A total of 69 chemical compounds were determined in five different PEOs. As the main chemical compounds, Cuminaldehyde was detected in cumin PEO, Sesquithujene was found in ginger PEO, and Eucalyptol (1,8-cineole) was determined in black cumin, chamomile, and cedarwood PEOs. All five PEOs were found to inhibit the growth of Alternaria species in a dose-dependent manner, whereas cumin EO was determined more inhibitory effect against A. solani and A. alternata. Cumin PEO showed the highest effect against Alternaria species because it contains a Cuminaldehyde chemical compound. The lowest inhibition percentage was found in chamomile PEO compared to other PEOs. This study suggested that cumin PEO has the potential as an antifungal agent for controlling of Alternaria diseases.

Molecular and chemical evaluation of patulin production of Aspergillus and Penicillium-like species isolated from Hungarian apples

Mycotoxins are secondary fungal metabolites harmful to humans and animals. Patulin (PAT) is a toxin found in different food products but especially in apples and their derivative products. The most common fungi producers of this compound are Aspergillus clavatus and Penicillium expansum. The production of patulin, as other mycotoxins, can be impacted by diverse phenomena such as water and nutrient availability, UV exposure, and the presence of antagonistic organisms. Consequently, gaining a comprehensive understanding of climate and environmental conditions is a crucial step in combating patulin contamination. In this study, moulds were isolated from 40 apple samples collected from seven locations across Hungary: Csenger, Damak, Pallag, Lövőpetri, Nagykálló, and Újfehértó. A total of 183 moulds were morphologically identified, with 67 isolates belonging to the Alternaria, 45 to the Aspergillus, and 13 to the Penicillium groups. The location possessed a higher influence than farming method on the distribution of mould genera. Despite the requirement of higher temperature, Aspergillus species dominated only for the region of Újfehértó with approximately 50% of the isolates belonging to the genus. Four of the seven locations assessed: Csenger, Debrecen-Pallag, Nyírtass and Nagykálló, were dominated by Alternaria species. All isolates belonging to the genera Aspergillus and Penicillium were tested for the presence of the isoepoxidone dehydrogenase (idh) gene, a key player in the patulin metabolic pathway. To guarantee patulin production, this ability was confirmed with TLC assays. The only Aspergillus strain that presented a positive result was the strain Aspergillus clavatus B9/6, originated from the apple cultivar Golden Reinders grown in Debrecen-Pallag by integrated farming. Of the Penicillium isolates only one strain, B10/6, presented a band of the right size (500-600 bp) for the idh gene. Further sequencing of the ITS gene showed that this strain should be classified as Talaromyces pinophilus. The TLC tests confirmed this microorganism as the only patulin producer under the studied conditions for its cluster.

Development and validation of stable isotope dilution LC–MS/MS method for simultaneous quantification of four Alternaria toxins in 15 food commodities

Alternaria toxins (ATs) are produced from Alternaria species that result in crop losses and harmful impacts on human health. A stable isotope dilution LC–MS/MS method was established to quantify four ATs in 15 food commodities: alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN), and tenuazonic acid (TeA). Based on systematically optimization of detection conditions and pre-processing steps, the limits of detection and limits of quantification of the four ATs ranged from 0.1 to 10 μg/kg and 0.2 to 30 μg/kg, respectively. The results showed that the recoveries of the four ATs were 72.0%–119.1%. The intra-precision and inter-precision ranged from 0.7% to 11.1% and 1.1% to 13.1%, respectively. The method was successfully applied to the determination of four ATs in 35 food samples, suggesting that this method could provide meaningful occurrence data to support the assessment of emerging ATs in food commodities.

First Report of Alternaria alternata Causing Leaf Spot on Catalpa bungei in China.

Catalpa bungei originates from China. It is fast-growing and possesses a vertically aligned trunk, rendering it a commendable construction material and a significant economic species. In July 2022, a serious leaf spot occurred in the LanLake farm (surveyed area of about 700 acres) in Nanyang (33°3'23" N, 112°28'50" E), Henan Province, China. The incidence rate of leaf disease reached 54% (n=100). The disease initially manifested as irregular round spots with a yellowish-brown hue, subsequently extending in all directions. Later, the lesion periphery exhibited a darkening effect, leading to yellowing. Twenty diseased leaves were randomly collected and cut into small pieces at the interfaces between infected and healthy tissues. The tissues were sterilized in a solution of 75% ethanol and 1% NaClO for 30 seconds and 1 minute, respectively. After rinsing in sterile water, the pieces were placed on potato dextrose agar (PDA) plates and incubated at 25°C for 5 days. A total of 29 purified fungal strains were acquired, exhibiting comparable phenotypes in terms of morphological characteristics. Three strains (QS1-1, QS1-2, and QS1-3) were isolated for subsequent investigations. The colony exhibited abundant aerial mycelium with shades ranging from dark green to grey-brown on the reverse side. To analyze the morphological characteristics of conidia, potato carrot agar (PCA) was used as the culture medium and incubated at 25°C with a 12-hour light/dark cycle. Conidia were obclavate or spheroidal, dark brown, with 3 to 5 transverse septa, and 1 to 4 longitudinal septa, measuring 12.4 to 36.7 × 4.4 to 9.0 μm (n=100), with conical beak lengths ranging from 0 to 4.3 μm. These morphological traits suggested that the pathogen shares similarities with the Alternaria species. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-alpha gene (tef1), glyceraldehyde 3-phosphate dehydrogenase gene (gapdh), and RNA polymerase II second largest subunit (rpb2) were amplified for further molecular identification. The resultant sequences were submitted to GenBank with the following accession numbers: OR733559, OR742124, OR761873 (ITS), OR939796, OR939797, OR939798 (tef1), OR939801, OR939802, OR939803 (gapdh), and PP054846, PP054847, PP054848 (rpb2). A Phylogenetic tree was constructed of combined genes (ITS, tef1, gapdh, and rpb2) of sequences, alongside the sequences of the type strains by the neighbor-joining method. The three strains formed a clade with the strains CBS 121456 of Alternaria alternata in phylogenetic trees, being separated from other Alternaria spp. The morphological features and molecular analyses supported the strains as members of Alternaria alternata (Woudenberg et al. 2015). To validate pathogenicity, a conidial suspension (106 conidia ml-1) of all three strains was inoculated onto three healthy leaves of five seedlings, with 50 μl of inoculum absorbed with cotton balls. Another group of five plants received sterile water as a control. All plants were incubated in a climate chamber at 28°C and 90% relative humidity. Four days post-inoculation, lesions resembling natural phenomena were observed, whereas control plants showed no symptoms. Subsequent reisolation produced cultures that were morphologically and molecularly identical to the original strains, fulfilling Koch's postulates. Stem canker of C. bungei caused by Phytophthora nicotianae has been reported in China (Chang et al. 2022). This is the first report of A. alternata causing leaf spots on C. bungei in China. Further research is required on management options to control this disease and the host range still needs to be clarified for accurate disease management.