First report of Stemphylium eturmiunum causing leaf spot and black spot on apple in Zhaotong, Yunnan, China

Apple is the largest fruit tree crop in the world, and China is the largest apple-producing County in the world. Zhaotong, Yunnan Province is a typical cold and mountainous apple-producing area in China. However, apple production is threatened by diseases during the entire growing season, and among them, apple leaf spot and fruit black spot are severe. In previous reports, the main pathogen causing apple leaf spot and fruit black spot was Alternaria sp. (Lior, et al, 2017), while different pathogens were identified. In the current study, seven red Fuji apple fruit with typical black spot samples were collected randomly in Dongda company orchard, Sujiayuan town, Zhaotong, Yunnan on March 25, 2021. The spots on the surface of these apples appear rounded, the diseased parts turn brown or black in colour and the flesh became soften and rotten. The tissues of fruit epidermis at the edge between diseased and healthy parts were cut, soaked in 75% alcohol for 30 s, washed with sterile water three times, and air-dried. Five pieces of tissue were placed on PDA medium amended with rifampicin (50 mg/ml) and incubated in the dark at 25 ℃ for 3-5 days. After colonies grew, mycelial clumps were picked out from the edges of the colonies, transferred to new PDA plates, and incubated at 25 ℃ for 6 days. The diameter of the colonies reached up to 5.7 cm. A representative isolate was retained for further work and was named P6-3-1. The hyphae were white and dense at an early stage, the culture medium on the underside became yellow and the middle parts of the colonies were darker. With maturity, hyphae were clumped, became red with other colors interspersed, and the medium became dark red. Light brown spores were produced, with more vertical septa and fewer transverse septa. Two to three transverse septa were generally observed with obvious constriction at the transverse septa. Average spore size was 22.83 µm ± 2.04 µm × 14.58 µm ± 1.97 µm. DNA was extracted from mycelium, purified and amplified with two pairs of primers, ITS1/ITS4 (White et al. 1990) and gpdF/gpdR (Marcos P. S. Câmara, et al. 2002). The PCR products were sequenced and deposited in GenBank (accession NO.OK560128 and OK627661 ). The similarity of ITS sequences between the isolate and MH843733 (Stemphylium eturmiunum strain ST14) was 100%, and that of gpd sequences between the isolate and MH843728 (Stemphylium eturmiunum strain ST20) was 100%. The maximum parsimony method of Mega7.0 was used and demonstrated that the studied isolate converged to the same branch as Stemphylium eturmiunum. Koch's postulates was applied to identify the pathogenicity of this isolate. A disc of P6-3-1-culture on PDA (5 mm in diameter) was placed on apple leaves and fruit wounds. Sterile PDA was used as a control. All plants were kept in a growth chamber at 25-30 ℃. Four days after inoculation, the disease spot was observed on the inoculated sites and fruit, and with the extension of incubation time, the diseased spots continue to grow, and the leaf spots were not limited by the veins. The pathogen was re-isolated from the inoculated leaves and fruit, satisfying Koch’s postulates. This pathogen can also cause postharvest rot of sweet cherry (Alice Spadoni, et al, 2020), postharvest rot on tomato (Prencipe Simona, et al, 2021), etc. This is the first report that Stemphylium eturmiunum can cause apple leaf spot and fruit black spot in Yunnan province, China. The apple black spot caused by Stemphylium eturmiunum was accurately identified. By distinguishing between the two similar diseases mentioned above, resistance to the host and management practices can be accrued based on the characteristics of the pathogen, its epidemiological pattern and the choice of an effective chemical fungicide.

Characterisation and Pathogenicity of Aspergillus tamarii Causing Banana Fruit Rot

Banana fruit rot is a common postharvest disease of the banana fruit. The appearance of rot symptoms on the surface of the fruits reduces the quality and marketability of banana. From rot lesions on banana fruits, three Aspergillus isolates were isolated. Based on morphological characteristics and sequences of Internal Transcribed Spacer, β-tubulin and calmodulin, the isolates were identified as A. tamarii. Pathogenicity tests of the isolates, conducted using mycelial plugs with wounded and unwounded treatments, showed A. tamarii as the pathogen of banana fruit rot. Rot symptoms were highly severe on wounded banana fruits compared to unwounded fruits, and therefore, wounded banana fruits are more susceptible to A. tamarii infection. To the best of our knowledge, this is the first report of A. tamarii as a causal pathogen of banana fruit rot. This study indicated A. tamarii is one of postharvest rot pathogens of banana.

Susceptibility of rosaceous pome and stone fruits to postharvest rot by Paecilomyces niveus

Paecilomyces rot of apples is a postharvest disease caused by Paecilomyces niveus, a problematic spoiling agent of fruit juices and derivatives. Processing fruits infected with Paecilomyces rot can lead to juices contaminated with P. niveus ascospores. These ascospores are heat-resistant and may survive food processing and germinate in finished products. Because the fungus produces the mycotoxin patulin, juice spoilage by P. niveus is an important health hazard. Little is known about the disease biology and control mechanisms of this recently described postharvest disease. The range of fruit products contaminated by P. niveus and patulin led us to hypothesize that the host range of Paecilomyces rot is broader than previously thought. Following Koch’s postulates, we determined that multiple untested rosaceous fruits and popular apple cultivars are susceptible to Paecilomyces rot infection, and that these infected fruits contain significant levels of patulin. We also observed that two closely related food spoiling fungi, Paecilomyces fulvus and Paecilomyces variotti, were unable to infect, cause symptoms in, or grow in wounded fruits. Hence, we challenge the assumption that P. niveus spoilage inoculum is introduced to foods solely through environmental sources, and show that other economically important rosaceous fruits, peaches, pears, sweet cherries and sour cherries, are susceptible to infection and can also serve as sources of spoilage inoculum. Our results highlight the unique abilities of Paecilomyces niveus to infect a variety of fruits, produce patulin, and form resistant spores capable of spoiling normally shelf-stable products.

First Report of Stemphylium eturmiunum causing postharvest rot on tomato (Solanum lycopersicum) in Italy

Italy is the largest tomato (Solanum lycopersicum)-producing country in Europe with a cultivated area of 97,092 ha and a production of 5,798,103 tons/year in 2018 (FAOSTAT, 2020). During July 2020, a postharvest rot occurred in fresh tomatoes ‘Piccadilly’ cultivated in Sicily (Pachino, RG) and commercialized in Northern Italy (Torino, TO). Affected fruit showed circular black rot on the blossom end. The rot had an average incidence of 7% of the fruits, in three batches of 100 tomatoes each. Isolation was carried out by cutting pieces of symptomatic rotten fruits. The fragments were surface-disinfected with 1% sodium hypochlorite for 30 s, rinsed in sterile water and air-dried. Five fragments were cut and plated onto Potato Dextrose Agar (PDA) supplemented with streptomycin, and incubated at 24±1°C in the dark for 5 days. Representative colonies were transferred onto PCA and morphological observations were performed as described by Woudenberg et al. (2017) after 7 and 14 days. Colonies were olive-green, flat with regular margins, while conidia were mid to deep brown, solitary, ovoid or ellipsoid (17.39 µm ± 2.04 × 10.59 ± 3.30 µm) with transverse and longitudinal septa. Based on morphological observations the isolates were identified as Stemphylium eturmiunum (Simmons, 2001). Species identification was confirmed by sequencing rDNA internal transcribed spacer (ITS) using primers ITS1/ITS4 (White et al. 1990), cmdA gene region using primers CALDF1/CALDR2 (Lawrence et al. 2013) and gapdh gene region with primers gpd1/gpd2 (Berbee et al. 1999). Six amplified sequences per region (ANos. from MW158387 to MW158398 and from MW159746 to MW159751) were BLAST-searched in GenBank, obtaining >99 % identity with ex-type strain of S. eturmiunum strain CBS 109845 (AN° KU850541) for ITS, and 100% identity (ANos. KU850831 and KU850689) for cmdA and gapdh, respectively. To confirm the species, DNA sequences were aligned with CLUSTAL W with closely related species of Stemphylium reported in the last revision of the genus (Woudenberg et al., 2017), and a phylogenetic analysis with the Neighbor Joining method based on Tamura Nei model + Gamma distribution (bootstrap 1,000) was performed. The phylogenetic tree confirmed the identity of the isolates as S. eturmiunum (Suppl. Fig. 1). To fulfil Koch’s postulates, pathogenicity tests were conducted on S. lycopersicum cv. Piccadilly fruits. Tomatoes were surface sterilized with 1% sodium hypochlorite and air-dried. Fruits (5 fruits per isolates) were wounded (two injuries of 3 mm each) and inoculated with a spore suspension of 1x105 cell/mL obtained from 15 days-old PCA cultures, as in Spadoni et al. (2020. Negative controls were wounded and inoculated with sterile deionized water. Symptoms occurred on all fruits inoculated after 12 days at 24±1°C and S. eturmiunum was re-isolated from inoculated fruits on PCA (Suppl. Fig. 2), control remained symptomless. Re-isolated colonies were molecularly identified as S. eturmiunum. In Italy a different species, S. vesicarium, was reported on tomato (Porta-Puglia, 1981), while S. eturmiunum was described as a postharvest pathogen of tomato in China, Greece, New Zealand and the United States (Woudenberg et al., 2017; Vaghefi et al., 2020), and from fruits commercialized in Danish and Spanish markets (Andersen and Frisvad, 2004). To the best of our knowledge, this is the first report of S. eturmiunum causing postharvest rot on tomato in Italy. The occurrence of this pathogen further stresses the importance of careful handling to prevent fruit crackings and of preharvest control strategies.

Susceptibility of rosaceous fruits and apple cultivars to postharvest rot by Paecilomyces niveus

Paecilomyces rot of apples is a postharvest disease caused by Paecilomyces niveus, a problematic spoiling agent of fruit juices and derivatives. The fungus produces ascospores that can survive food processing and germinate in finished fruit products. Processing apple fruits infected with Paecilomyces rot can lead to P. niveus contaminated juices. Because the fungus produces the mycotoxin patulin, juice spoilage by P. niveus is an important health hazard. Little is known about the disease biology and control mechanisms of this recently described postharvest disease. Following Kochs postulates, we determined that a range of previously untested rosaceous fruits and popular apple cultivars are susceptible to Paecilomyces rot infection. We also observed that two closely related food spoiling fungi, Paecilomyces fulvus and Paecilomyces variotti, were unable to infect, cause symptoms in, or reproduce in wounded fruits. Our results highlight the unique abilities of Paecilomyces niveus to infect a variety of fruits, produce patulin, and form highly-resistant spores capable of spoiling normally shelf-stable products.

Morphological and molecular characterization of Alternaria spp. isolated from European pears

In a recent survey of postharvest rot pathogens in European pear in Southern Oregon, Alternaria spp. were frequently isolated from orchard samples of pear flowers and fruits. Morphological differences were observed within the isolated cultures. A preliminary NCBI BLAST search analysis using sequences of the ATPase locus across 94 isolates of Alternaria spp. obtained from pear fruit rots, revealed three major Alternaria sections, sect. Alternata, sect. Infectoriae, and sect. Ulocladioides. Thirteen isolates were selected based on their genetic and morphological diversity across three Alternaria sections and were subjected to multilocus phylogenetic analysis using sequences from plasma membrane ATPase, calmodulin, and Alt a1 loci. Within section Alternata, four A. arborescens isolates and one A. destruens isolate were identified; within sections Infectoriae and Ulocladioides, one A. rosae isolate and two A. botrytis isolates were identified, respectively. The remaining five isolates could not be identified based on the available sequences for the three loci used in this study. In addition to the phylogenetic analysis, pathogenicity assays revealed differential responses to these isolates on four pear cultivars Anjou, Bartlett, Comice, and Bosc. Inoculation of isolates within Alternaria sect. Alternata resulted in fruit lesions across all cultivars with Bosc being significantly susceptible (p<0.0001). Isolates within Alternaria sect. Ulocladioides caused rots on Anjou and Bosc, while isolates within Alternaria sect. Infectoriae developed rots on Bosc only. This study suggests that there is differential susceptibility of pear cultivars to Alternaria rots and the severity of postharvest rot depends on the type of Alternaria spp. and cultivar predominant in a region.