First Report of Colletotrichum chrysophilum Causing Apple Bitter Rot in Spain

Bitter rot of apple (Malus × domestica Borkh.) is a cosmopolitan disease affecting fruit and causes considerable losses worldwide. In September 2020, symptoms of bitter rot were observed on ‘Pink Lady’ apples in two orchards (~2.5 ha each) in Gualta, Catalonia, Spain (42.03803 N, 3.09831 E, and 42.03942 N, 3.10931 E). Early symptoms consisted of light brown and sunken circular lesions (1-4 mm) that enlarged over time, later becoming dark brown and water soaked, and extending cone-shaped toward the core. Sporulation was mostly noticed in larger lesions. Estimated incidence was 2% and 20% of 150 trees surveyed in each orchard, respectively. Twenty-one fungal isolates were obtained from diseased fruit by culturing small pieces of necrotic tissue on potato dextrose agar (PDA) amended with rifampicin at 50 mg/liter. Colonies on PDA looked identical. They were cottony, initially light-gray colored on top and darkening with age; colony reverse initially cream colored and darkening with age. Conidia were produced in orange acervular masses on Spezieller Nährstoffarmer Agar, and were aseptate, hyaline, cylindrical with obtuse ends, and measuring 10.1 to 14.7 × 4.5 to 7.1 μm (average 13.1 ± 1.04 × 5.3 ± 0.67 μm [mean ± SD], n = 50), with a mean length/width ratio 2.6 ± 0.39 (n = 16 isolates). Perithecia were not observed. Based on the conidial morphology, the isolates were tentatively identified as belonging to the Colletotrichum gloeosporioides species complex (Weir et al. 2012). Total genomic DNA was extracted from all isolates and six nuclear regions were amplified and partially sequenced: the internal transcribed spacer region of rDNA (ITS), the mating type protein 1-2-1 gene and the Mat1-2-1-Apn2 intergenic spacer region (ApMAT), actin (ACT), calmodulin (CAL), glyceraldehyde 3-P dehydrogenase (GAPDH), and tubulin (TUB2). The sequences for each region were 100% identical across all isolates. BLAST searches in GenBank showed 99-100% identity with sequences of various C. chrysophilum W.A.S. Vieira, W.G. Lima, M.P.S. Câmara & V.P. Doyle strains including the ex-type CMM4268 (Vieira et al. 2017). Sequences of the representative isolate CJL1080 were deposited in GenBank (ACT, MZ488944; ApMAT, MZ442299; CAL, MZ488945; GAPDH, MZ488946; ITS, MZ443972; TUB2, MZ442300). A multilocus phylogenetic analysis through Bayesian inference conducted with the obtained sequences and reference ones (Khodadadi et al. 2020) revealed that our isolates clustered well within C. chrysophilum, as suggested by BLAST results. To confirm Koch’s postulates, isolates CJL1080 and CJL1095 were inoculated on ‘Pink Lady’ apples. Six surface-sterilized fruits per isolate were wound-inoculated four times each with either 20 μl of a conidial suspension (105 conidia/ml) or sterile distilled water (control). After 7 days of incubation in a moist chamber at 22°C, symptoms compatible with Colletotrichum infection were observed around the wounds, whereas control inoculations remained symptomless. The fungus was reisolated from all the lesions and identified through its morphological traits and DNA sequencing (ApMat, CAL, and GAPDH). No fungus was isolated from the controls. Taxa of the C. gloeosporioides species complex causing bitter rot have been recently reported in Europe (Grammen et al. 2019; Nodet et al. 2019). This is the first report of C. chrysophilum causing apple bitter rot in Spain, which expands the knowledge on the geographic distribution of this important pathogen of apple in Europe.

The decay and fungal succession of apples with bitter rot across a vegetation diversity gradient

Bitter rot is a disease of apple caused by fungi in the genus Colletotrichum. Management begins with removal of infected twigs and fruits from tree canopies to reduce overwintering inoculum. Infected apples are usually tossed to the orchard floor, which is generally managed as herbicide-treated weed-free tree rows, separated by grass drive rows. We monitored decay rates and succession of fungi of apples with bitter rot in tree canopies, and on the soil surface in tree rows, grass drive rows, and nearby diverse plant communities. We hypothesized that decay would occur most rapidly within diverse plant communities, which would provide a more diverse array of potential fungal decomposers. Apples in tree canopies became dry and mummified and had more Colletotrichum gene marker copies the following growing season than did apples on the soil surface. Of the soil surface samples, those in grass drive rows and diverse plant communities had higher moisture, faster decay rates, and sharper decreases in Colletotrichum gene marker copies than apples in tree rows. Fungal composition across all decaying apples was dominated by yeasts, with higher genus-level richness, diversity, and evenness in apples from tree canopies than those on the soil surface. In soil surface apples, we observed clear successional waves of Pichia, Kregervanrija, and [Candida] yeasts, with similar but distinctly diverging fungal composition. Our results show that orchard floor management can influence fungal succession in apples with bitter rot, but suggests that bitter rot management should primarily focus on removing infected apples from tree canopies.

Comparative transcriptome analysis reveals significant differences in gene expression between pathogens of apple Glomerella leaf spot and apple bitter rot

Background: Apple Glomerella leaf spot (GLS) and apple bitter rot (ABR) are two devastating foliar and fruit diseases on apple. The different symptoms of GLS and ABR could be related to different transcriptome patterns. Thus, the objectives of this study were to compare the transcriptome profiles of Colletotrichum gloeosporioides, the common pathogen of GLS and ABR, and to evaluate the genes involvement on pathogenicity.Results: A relatively large difference was discovered between the GLS- and ABR-isolate, and quite a number of differential expression genes associated with pathogenicity were revealed. The DEGs between the GLS- and ABR-isolate were significantly enriched in GO terms of secondary metabolites, however the categories of degradation of various cell wall components did not. A number of genes associate with secondary metabolism were revealed. A total of 17 Cytochrome P450s (CYP), 11 of which were up-regulated while six were down-regulated, and five up-regulated methyltransferase genes were discovered. The genes associated with secretion of extracellular enzymes and melanin accumulation were up-regulated. Four genes associated with degradation of host cell wall, three genes involved in degradation of cellulose, and one gene involved in degradation of xylan were revealed and all up-regulated. In addition, genes involved in melanin synthesis, such as tyrosinase and glucosyltransferase, were highly up-regulated.Conclusions: The penetration ability, pathogenicity of GLS-isolate was greater than that ABR-isolate, which might be indicate that GLS-isolate originated from ABR-isolates by mutation. These results contributed to highlight the importance to investigate such DEGs between GLS- and ABR-isolate in depth.

Fungicide sensitivity of Colletotrichum species causing bitter rot of apple in the Mid-Atlantic United States

Apple growers in the Mid-Atlantic region of the United States have reported increased losses to bitter rot of apple. We tested the hypothesis that this increase is because the Colletotrichum population has developed resistance to commonly used single-mode-of-action (single-MoA) fungicides. We screened 220 Colletotrichum isolates obtained from 38 apple orchards in the Mid-Atlantic region for resistance to 11 fungicides in FRAC (Fungicide Resistance Action Committee) groups 1, 7, 9, 11, 12, and 29. Eleven (5%) of these isolates were resistant to FRAC group 1 with confirmed beta-tubulin E198A mutations, and two (< 1%) were also resistant to FRAC group 11 with confirmed cytochrome-b G143A mutations. Such low frequencies of resistant isolates indicate that fungicide resistance is unlikely to be the cause of any regional increase in bitter rot. A subsample of isolates was subsequently tested in vitro for sensitivity to every single-MoA fungicide registered for apple in the Mid-Atlantic US (22 fungicides; FRAC groups 1, 3, 7, 9, 11, 12, and 29), and thirteen fungicides were tested in field trials. These fungicides varied widely in efficacy both within and between FRAC groups. Comparisons of results from our in vitro tests with results from our field trials and other field trials conducted across the Eastern US suggested that EC₂₅ values (concentrations that reduce growth by 25%) are better predictors of fungicide efficacy in normal field conditions than EC₅₀ values. We present these results as a guideline for choosing single-MoA fungicides for bitter rot control in the Mid-Atlantic US.

Colletotrichum fioriniae and C. godetiae causing postharvest bitter rot of apple in South Tyrol (northern Italy)

South Tyrol (northern Italy) harbors one of the largest interconnected apple farming areas in Europe that contributes approximately 10% to the apple production of the European Union. In spite of the availability of sophisticated storage facilities, postharvest diseases occur, one of which is bitter rot of apple. In Europe, this postharvest disease is mainly caused by the Colletotrichum acutatum species complex. This work aimed to characterize the Colletotrichum species isolated from decayed apple fruit collected in 2018 and 2019 in South Tyrol. The characterization of Colletotrichum species was accomplished based on multi-locus DNA sequences of four different genomic regions, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3), and the internal transcribed spacer (ITS) region as well as morphological and pathogenicity assessment. A phylogenetic analysis based on multi-locus DNA sequences showed that the isolates obtained from apples with symptoms of bitter rot belonged to the species C. godetiae and C. fioriniae, which are part of the C. acutatum species complex. A third species isolated from apple belonging to the same species complex, C. salicis, was recently described in this area. Moreover, the Colletotrichum isolates found in this study proved to be virulent on the cultivars ‘Cripps Pink’, ‘Golden Delicious’ and ‘Roho 3615’/Evelina®. To the best of our knowledge, C. godetiae and C. fioriniae have so far never been mentioned as postharvest pathogens of apple in Italy, even though the (re)-analysis of samples collected in the past indicates that these pathogens have been occurring in Italy for at least a decade. So far, bitter rot seems to play a rather minor role as a postharvest disease in South Tyrol, but it was disproportionately represented on few scab-resistant apple cultivars, which are increasingly planted in organically managed orchards. Considering that the expansion of organic apple production and the conversion to new potentially Colletotrichum-susceptible cultivars will continue, the present study represents a first important contribution towards a better understanding of bitter rot in this geographic area.

Bitter Rot of Apple in the Mid-Atlantic US: Causal Species and Evaluation of the Impacts of Regional Weather Patterns and Cultivar Susceptibility

Apple growers in the Mid-Atlantic region of the United States have been reporting an increase in losses to bitter rot of apple and are requesting up-to-date management recommendations. Management is complicated by variations in apple cultivar susceptibility, temperature and rainfall, and biology of the Colletotrichum species that cause bitter rot. Over 500 apples with bitter rot were obtained from 38 orchards across the Mid-Atlantic and the causal species identified as C. fioriniae and C. nymphaeae of the C. acutatum species complex and C. chrysophilum, C. noveboracense, C. siamense, C. fructicola, C. henanense, and C. gloeosporioides sensu stricto of the C. gloeosporioides species complex, the latter two being first reports. Species with faster in vitro growth rates at higher temperatures were more abundant in warmer regions of the Mid-Atlantic, while those with slower growth rates at higher temperatures were more abundant in cooler regions. Regional bloom dates are earlier and weather data shows a gradual warming trend that likely influenced, but was not necessarily the main cause of the recent increase in bitter rot in the region. A grower survey of apple cultivar susceptibility showed high variation, with the increase in acres planted to the highly susceptible cultivar ‘Honeycrisp’ broadly corresponding to the increase in reports of bitter rot. These results form a basis for future studies on the biology and ecology of the Colletotrichum species responsible, and suggest that integrated bitter rot management must begin with selection of less-susceptible apple cultivars.

Characterization of Bacillus velezensis AK-0 as a biocontrol agent against apple bitter rot caused by Colletotrichum gloeosporioides

Bacillus genus produces several secondary metabolites with biocontrol ability against various phytopathogens. Bacillus velezensis AK-0 (AK-0), an antagonistic strain isolated from Korean ginseng rhizospheric soil, was found to exhibit antagonistic activity against several phytopathogens. To further display the genetic mechanism of the biocontrol traits of AK-0, we report the complete genome sequence of AK-0 and compared it with complete genome sequences of closely related strains. We report the biocontrol activity of AK-0 against apple bitter rot caused by Colletotrichum gloeosporioides, which could lead to commercialization of this strain as a microbial biopesticide in Korea. To retain its biocontrol efficacy for a longer period, AK-0 has been formulated with ingredients for commercialization, named AK-0 product formulation (AK-0PF). AK-0PF played a role in the suppression of the mycelial growth of the fungicide-resistant pathogen C. gloeosporioides YCHH4 at a greater level than the non-treated control. Moreover, AK-0PF exhibited greater disease suppression of bitter rot in matured under field conditions. Here, we report the complete genome sequence of the AK-0 strain, which has a 3,969,429 bp circular chromosome with 3808 genes and a G+C content of 46.5%. The genome sequence of AK-0 provides a greater understanding of the Bacillus species, which displays biocontrol activity via secondary metabolites. The genome has eight potential secondary metabolite biosynthetic clusters, among which, ituD and bacD genes were expressed at a greater level than other genes. This work provides a better understanding of the strain AK-0, as an effective biocontrol agent (BCA) against phytopathogens, including bitter rot in apple.

First report of preharvest fruit rot of ‘Pink Lady’ apples caused by Colletotrichum fructicola in Italy

In late summer 2019, a severe outbreak of fruit rot was observed in commercial ‘Pink Lady’ apple orchards (>20 ha in total) in the region Emilia-Romagna (Northern Italy). The symptoms on the fruit appeared as small circular red to brown lesions. Disease incidences of over 50% of the fruits were observed. To isolate the causal agent, 15 affected apples were collected and small portions of fruit flesh were excised from the lesion margin and placed on potato dextrose agar (PDA). The plates were incubated at 20°C in the dark, and pure cultures were obtained by transferring hyphal tips on PDA. The cultures showed light to dark gray, cottony mycelium, with the underside of the culture being brownish and becoming black with age. Conidia (n=20) were cylindrical, aseptate, hyaline, rounded at both ends, and 12.5 to 20.0 × 5.0 to 7.5 μm. The morphological characteristics were consistent with descriptions of Colletotrichum species of the C. gloeosporioides species complex, including C. fructicola (Weir et al. 2012). The identity of two representative isolates (PinkL2 & PinkL3) from different apples was confirmed by means of multi-locus gene sequencing. Genomic DNA was extracted using the LGC Mag Plant Kit (Berlin, Germany) in combination with the Kingfisher method (Waltham, USA). Molecular identification was conducted by sequencing the ITS1/ITS4 region and partial sequences of four other gene regions: chitin synthase (CHS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and beta-tubulin (TUB). The sequences have been deposited in GenBank under accession numbers MT421924 & MT424894 (ITS), MT424612 & MT424613 (CHS), MT424616 & MT424617 (GAPDH), MT424614 & MT424615 (ACT), and MT424620 & MT424621 (TUB). MegaBLAST analysis revealed that our ITS sequences matched with 100% identity to Colletotrichum fructicola (Genbank JX010177). The CHS, GAPDH, ACT and TUB sequences of both isolates were 100% identical with C. fructicola culture collection sequences in Genbank (JX009807, JX009923, JX009436 and JX010400, respectively), confirming the identity of these isolates as C. fructicola. Koch's postulates were performed with 10 mature ‘Pink Lady’ apples. Surface sterilized fruit were inoculated with 20 μl of a suspension of 105 conidia ml–1 after wounding with a needle. The fruits were incubated at 20˚C at high relative humidity. Typical symptoms appeared within 4 days on all fruit. Mock-inoculated controls with sterile water remained symptomless. The fungus was reisolated and confirmed as C. fructicola by morphology and sequencing of all previously used genes. Until recently the reported causal agents of bitter rot of apple in Europe belong to the Colletotrichum acutatum species complex (Grammen et al. 2019). C. fructicola, belonging to C. gloeosporioides species complex, is known to cause bitter rot of apple in the USA, Korea, Brazil, and Uruguay (Kim et al. 2018; Velho et al. 2015). There is only one report of bitter rot associated with C. fructicola on apple in Europe (France) (Nodet et al. 2019). However, C. fructicola is also the potential agent of Glomerella leaf spot (GLS) of apple (Velho et al. 2015; 2019). To the best of our knowledge this is the first report of C. fructicola on apples in Italy. It is important to stress that the C. gloeosporioides species complex is still being resolved and new species on apple continue to be identified, e.g. C. chrysophilum that is very closely related to C. fructicola (Khodadadi et al. 2020). Given the risks of this pathogen the presence of C. fructicola in European apple orchards should be assessed and management strategies developed.