Phylogenetic Diversity and Phenotypic Characterization of Phlyctema vagabunda (syn. Neofabraea alba) and Neofabraea kienholzii Causing Postharvest Bull's Eye Rot of Apple in Northern Italy.

Bull's eye rot, caused by Phlyctema vagabunda and Neofabraea species, is one of the most important postharvest diseases of apple. South Tyrol (northern Italy) is the largest continuous apple-producing area in Europe, with approximately 1 million tons being produced yearly and conserved in technologically advanced storage facilities for several months. Still, studies on the pathogen species causing postharvest bull's eye rot of apple, as well as their diversity and biology, are lacking for this region. Therefore, the main purpose of the present work was to identify and characterize fungal isolates obtained from decayed apple fruit with symptoms of bull's eye rot that were collected in 2018 and 2019 in different packinghouses in South Tyrol. Among more than 1,000 fungal isolates that were obtained, 419 could be assigned to the genera Phlyctema and/or Neofabraea based on rot symptoms on apple fruit and colony morphology on potato dextrose agar. A smaller subset of 101 representative isolates was further analyzed by DNA sequencing of the internal transcribed spacer region. Furthermore, partial segments of the ß-tubulin gene, the translation elongation factor 1α gene, and the 16S mitochondrial ribosomal RNA gene were studied. The phylogenetic analyses, including sequences of reference species, showed that P. vagabunda is the dominant species associated with bull's eye rot of apple in the study area, whereas Neofabraea kienholzii was found only on a small number of apple fruit samples. The combination of multilocus sequence data revealed 11 unique genotypes that belonged to P. vagabunda and four to N. kienholzii. To the best of our knowledge, this study is the first to report N. kienholzii as a postharvest pathogen of apple in Italy. Finally, a pathogenicity test demonstrated different degrees of virulence among selected isolates of P. vagabunda and N. kienholzii on the cultivar Golden Delicious. The present study emphasizes the importance of accurate species identification, because different species may vary in their biological and pathogenic characteristics, and consequently require distinct disease management strategies, both in the field and during the postharvest stages.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Colletotrichum fioriniae and Colletotrichum 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, contributing approximately 10% to the apple production of the European Union. Despite 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 study aimed to characterize the Colletotrichum spp. isolated from decayed apple fruit collected in 2018 and 2019 in South Tyrol. The characterization of Colletotrichum spp. was accomplished based on multilocus 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 multilocus DNA sequences showed that the isolates obtained from apples with symptoms of bitter rot belonged to the species Colletotrichum godetiae and Colletotrichum fioriniae, which are part of the Colletotrichum acutatum species complex. A third species isolated from apples belonging to the same species complex, Colletotrichum salicis, was described in this area. Moreover, the Colletotrichum isolates found in this study proved to be virulent on 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, although the reanalysis 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 minor role as a postharvest disease in South Tyrol, but it was disproportionately represented on a 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 an important contribution toward a better understanding of bitter rot in this geographic area.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

First Report of Colletotrichum salicis Causing Bitter Rot of Apple in Italy.

Apple (Malus domestica Borkh.) is the most important fruit crop in South Tyrol (northern Italy), with a total production of 905.089 tons in 2019 (Chamber of Commerce, Industry, Crafts and Agriculture of Bozen-Bolzano 2020). Symptoms of bitter rot were observed on organic apples of the cultivar 'Roho 3615'/Evelina® collected in a packinghouse in South Tyrol at the end of March 2018 after approximately six months of storage in controlled atmosphere. Lesions were circular and light brown with orange conidial masses. Tissue samples were removed under aseptic conditions from surface-cleaned fruit at the margin between healthy and diseased pulp tissue, transferred to Petri dishes with potato dextrose agar (PDA), and incubated in the dark at room temperature for two weeks. Single spore cultures were obtained by adapting the procedure of Choi et al. (1999). Pure cultures were grown in quadruplicate on PDA at 20°C in the dark for two weeks. The colony appearance on the upper side was mostly flat with a distinct margin, the surface was covered with short, floccose aerial mycelium, and the color ranged from light gray to dark gray, while the reverse side appeared yellow. From each replicate culture, conidia were harvested, and the length and width of 50 randomly selected conidia were measured using a compound light microscope coupled to a digital camera (Leica DMLS, Leica Microsystems, Wetzlar, Germany). Conidia were cylindrical to fusiform, pointed at one end, and measured 10.0 to 19.5 × 2.5 to 5.0 µm (14.5 ± 1.9 × 3.9 ± 0.7 µm [mean ± SD]) in consistency with Damm et al. (2012). In order to determine the species of isolate 18-DSS-BS-EL-1-012, a multi-locus DNA sequence analysis was performed. Genomic DNA was extracted by following the protocol described by Cassago et al. (2002). Four loci, actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone H3 (HIS3) and the internal transcribed spacer (ITS) region of the rRNA operon were amplified by PCR and Sanger sequenced (Damm et al. 2012; Lévesque and de Cock 2004). The obtained DNA sequences of ACT, GAPDH, HIS3 and ITS were 186, 150, 317 and 505 bp long and were submitted to GenBank under the accession numbers MT347599, MT347600, MT347598 and MT337388, respectively. A MegaBLAST analysis resulted in 100% sequence identity at all four loci with a type culture of Colletotrichum salicis (CBS 607.94; GenBank accession numbers: JQ949781, JQ948791, JQ949451 and JQ948460), which belongs to the C. acutatum species complex (Damm et al. 2012). A pathogenicity test was performed with twelve 'Golden Delicious' apples by wounding the fruit with a sterile piercing tool and inoculating 20 µl of spore suspension (104 conidia per ml) from a 21-day-old PDA culture. Inoculated fruits were incubated for 21 days in a moist chamber at 20°C in the dark. The symptoms were recorded at 3, 5, 7, 10, 14 and 21 days post-inoculation (dpi). Symptoms appeared after 7 days on all inoculated fruits and resembled those observed on the original fruit, while mock-inoculated controls with sterile water remained symptomless. Fungal colonies resembling the original culture were re-isolated from lesions on the apple and plated on PDA. Their identity was confirmed by DNA sequence analysis of the ITS region, thereby proving Koch's postulates. Bitter rot occurs globally and is considered one of the most important diseases of apple that has the potential to cause significant crop losses (Sutton et al. 2014). Other Colletotrichum species have been commonly reported from apple in Europe, such as C. godetiae and C. fioriniae, whereas C. salicis has been reported solely in New Zealand and Belgium (Damm et al. 2012; Grammen et al. 2019). To the best of our knowledge, this is the first report of C. salicis causing bitter rot of apple in Italy.

Callose and cellulose synthase gene expression analysis from the tight cluster to the full bloom stage and during early fruit development in Malus × domestica.

Apple (Malus × domestica) is an economically important temperate fruit-bearing crop which belongs to the family of Rosaceae and its pomaceous fruit is one of the most commonly cultivated. Several studies have demonstrated that the cell wall plays a pivotal role during flower and fruit development. It takes active part in pollen tube growth and contributes to determine the fruit firmness trait through the action of cell wall-related enzymes (i.e. polygalacturonase and pectinmethylesterase). We have investigated the expression of callose and cellulose synthase genes during flowering from tight cluster to anthesis and during early fruit development in domesticated apple. We also link the changes observed in gene expression to the profile of soluble non-structural carbohydrates at different developmental stages of flowers/fruitlets and to the qualitative results linked to wall polysaccharides' composition obtained through near-infrared spectroscopy. This work represents an important addition to the study of tree physiology with respect to the analysis of the expression of callose and cellulose synthase genes during flower and early fruit development in domesticated apple.

Molecular genetic variability of Cryphonectria hypovirus 1 associated with Cryphonectria parasitica in South Tyrol (northern Italy).

Cryphonectria hypovirus 1 (CHV-1) has been widely studied and used as a biocontrol agent because of its ability to infect the chestnut blight fungus, Cryphonectria parasitica, and to reduce its virulence. Knowledge about the hypovirus, its presence, and diversity is completely lacking in South Tyrol (northern Italy), which may obstruct biocontrol measures for chestnut blight based on CHV-1. This work aimed to study the occurrence of CHV-1 infecting C. parasitica in South Tyrol and to perform a genetic characterization of the hypovirus. In South Tyrol, CHV-1 was found to occur in 29.2% of the fungal isolates investigated, varying in frequency between different regions and chestnut stands. Twenty-three haplotypes based on partial cDNA (complementary DNA) sequences of open reading frame (ORF)-A and 30 haplotypes based on partial cDNA sequences of ORF-B were identified among 47 and 56 hypovirulent fungal isolates, respectively. Phylogenetic analysis showed that all the haplotypes belonged to the Italian subtype of CHV-1 and that they were closely related to the populations of Italy, Switzerland, Croatia and Slovenia. Evidence of recombination was not found in the sequences and point mutations were the main source of diversity. Overall, this study indicated that the prevalence of CHV-1 in South Tyrol is low compared to many other central and western European populations and determined a need to actively impose biocontrol measures. Using sequence analysis, we identified some variants of interest of CHV-1 that should be studied in detail for their potential use in biocontrol.

Heat shock transcriptional factors in Malus domestica: identification, classification and expression analysis.

BACKGROUND: Heat shock transcriptional factors (Hsfs) play a crucial role in plant responses to biotic and abiotic stress conditions and in plant growth and development. Apple (Malus domestica Borkh) is an economically important fruit tree whose genome has been fully sequenced. So far, no detailed characterization of the Hsf gene family is available for this crop plant. RESULTS: A genome-wide analysis was carried out in Malus domestica to identify heat shock transcriptional factor (Hsf) genes, named MdHsfs. Twenty five MdHsfs were identified and classified in three main groups (class A, B and C) according to the structural characteristics and to the phylogenetic comparison with Arabidopsis thaliana and Populus trichocarpa. Chromosomal duplications were analyzed and segmental duplications were shown to have occurred more frequently in the expansion of Hsf genes in the apple genome. Furthermore, MdHsfs transcripts were detected in several apple organs, and expression changes were observed by quantitative real-time PCR (qRT-PCR) analysis in developing flowers and fruits as well as in leaves, harvested from trees grown in the field and exposed to the naturally increased temperatures. CONCLUSIONS: The apple genome comprises 25 full length Hsf genes. The data obtained from this investigation contribute to a better understanding of the complexity of the Hsf gene family in apple, and provide the basis for further studies to dissect Hsf function during development as well as in response to environmental stimuli.

Analysis of cellulose synthase genes from domesticated apple identifies collinear genes WDR53 and CesA8A: partial co-expression, bicistronic mRNA, and alternative splicing of CESA8A.

Cellulose synthase (CesA) genes constitute a complex multigene family with six major phylogenetic clades in angiosperms. The recently sequenced genome of domestic apple, Malus×domestica, was mined for CesA genes, by blasting full-length cellulose synthase protein (CESA) sequences annotated in the apple genome against protein databases from the plant models Arabidopsis thaliana and Populus trichocarpa. Thirteen genes belonging to the six angiosperm CesA clades and coding for proteins with conserved residues typical of processive glycosyltransferases from family 2 were detected. Based on their phylogenetic relationship to Arabidopsis CESAs, as well as expression patterns, a nomenclature is proposed to facilitate further studies. Examination of their genomic organization revealed that MdCesA8-A is closely linked and co-oriented with WDR53, a gene coding for a WD40 repeat protein. The WDR53 and CesA8 genes display conserved collinearity in dicots and are partially co-expressed in the apple xylem. Interestingly, the presence of a bicistronic WDR53-CesA8A transcript was detected in phytoplasma-infected phloem tissues of apple. The bicistronic transcript contains a spliced intergenic sequence that is predicted to fold into hairpin structures typical of internal ribosome entry sites, suggesting its potential cap-independent translation. Surprisingly, the CesA8A cistron is alternatively spliced and lacks the zinc-binding domain. The possible roles of WDR53 and the alternatively spliced CESA8 variant during cellulose biosynthesis in M.×domestica are discussed.

Duplex TaqMan Real-Time PCR for Rapid Quantitative Analysis of a Phytoplasma in Its Host Plant without External Standard Curves.

The chapter describes a simple quantitative approach to assess phytoplasma load in samples obtained from "Candidatus Phytoplasma mali"-infected apple plants without the use of external standard curves. The assay is based on the simultaneous detection of a gene of the pathogen and a gene of the host plant in a duplex single-tube real-time PCR reaction using TaqMan chemistry. The quantity of the phytoplasma, relative to its host plant, is determined as the difference between the CT values of the two target genes (ΔCT). A critical data analysis step, affecting the inter-assay reproducibility between different amplification runs, is the setting of the threshold level, which is achieved by the recurrent analysis of a calibrator sample. The relative quantification procedure allows analyzing 45 DNA samples in duplicates on a 96-well reaction plate, in addition to the control and calibrator samples, and thus contributes to a substantial increase of analysis throughput and decrease of reagent/consumable costs per sample.

A new approach to apple proliferation detection: a highly sensitive real-time PCR assay.

The present paper describes a new approach for diagnosis of apple proliferation (AP) phytoplasma in plant material using a multiplex real-time PCR assay simultaneously amplifying a fragment of the pathogen 16S rRNA gene and the host, Malus domestica, chloroplast gene coding for tRNA leucine. For the first time, such an approach, with an internal analytical control, is described in a diagnostic procedure for plant pathogenic phytoplasmas enabling distinction between uninfected plant material and false-negative results caused by PCR inhibition. Pathogen detection is based on the highly conserved 16S rRNA gene to ensure amplification of different AP phytoplasma strains. The newly designed primer/probe set allows specific detection of all examined AP strains, without amplifying other fruit tree phytoplasmas or more distantly related phytoplasma strains. Apart from its specificity, real-time PCR with serial dilutions of initial template DNA ranging over almost five orders of magnitude (undiluted to 80,000-fold diluted) demonstrated linear amplification over the whole range, while conventional PCR showed a reliable detection only up to 500-fold or 10,000-fold dilutions, respectively. Compared to existing analytical diagnostic procedures for phytoplasmas, a rapid, highly specific and highly sensitive diagnostic method becomes now available.

Gene expression and biochemical changes of carbohydrate metabolism in in vitro micro-propagated apple plantlets infected by 'Candidatus Phytoplasma mali'.

'Candidatus Phytoplasma mali' (Ca. P. mali) is the disease agent causing apple proliferation (AP), which has detrimental effects on production in many apple growing areas of Central and Southern Europe. The present study investigated transcriptional and biochemical changes related to the sugar metabolism as well as expression of pathogenesis-related (PR) protein genes in in vitro micro-propagated AP-infected and healthy control plantlets with the aim of shedding light on host plant response to 'Ca. P. mali' infection. Expression changes between infected and control plantlets were detected by quantitative real-time PCR analysis. The most significant transcriptional changes were observed for genes coding for pathogenesis-related proteins and for heat shock protein 70, as well as for some genes related to the sugar metabolism, such as a sorbitol transporter (SOT5), hexokinase, sucrose-phosphate synthase or granule bound starch synthase. Furthermore, biochemical analyses revealed that infected plantlets were characterized by a significant accumulation of starch and sucrose, while hexoses, such as glucose and fructose, and sorbitol were present at lower concentrations. In summary, the present analysis provides an overview of a gene set that is involved in response to phytoplasma infection and, therefore, it may help for a better understanding of the molecular mechanisms involved in phytoplasma-host plant interaction in apple.

A gene expression analysis of cell wall biosynthetic genes in Malus x domestica infected by 'Candidatus Phytoplasma mali'.

Apple proliferation (AP) represents a serious threat to several fruit-growing areas and is responsible for great economic losses. Several studies have highlighted the key role played by the cell wall in response to pathogen attack. The existence of a cell wall integrity signaling pathway which senses perturbations in the cell wall architecture upon abiotic/biotic stresses and activates specific defence responses has been widely demonstrated in plants. More recently a role played by cell wall-related genes has also been reported in plants infected by phytoplasmas. With the aim of shedding light on the cell wall response to AP disease in the economically relevant fruit-tree Malus × domestica Borkh., we investigated the expression of the cellulose (CesA) and callose synthase (CalS) genes in different organs (i.e., leaves, roots and branch phloem) of healthy and infected symptomatic outdoor-grown trees, sampled over the course of two time points (i.e., spring and autumn 2011), as well as in in vitro micropropagated control and infected plantlets. A strong up-regulation in the expression of cell wall biosynthetic genes was recorded in roots from infected trees. Secondary cell wall CesAs showed up-regulation in the phloem tissue from branches of infected plants, while either a down-regulation of some genes or no major changes were observed in the leaves. Micropropagated plantlets also showed an increase in cell wall-related genes and constitute a useful system for a general assessment of gene expression analysis upon phytoplasma infection. Finally, we also report the presence of several 'knot'-like structures along the roots of infected apple trees and discuss the occurrence of this interesting phenotype in relation to the gene expression results and the modalities of phytoplasma diffusion.

Species-specific population structure in rock-specialized sympatric cichlid species in Lake Tanganyika, East Africa.

Species richness and geographical phenotypic variation in East African lacustrine cichlids are often correlated with ecological specializations and limited dispersal. This study compares mitochondrial and microsatellite genetic diversity and structure among three sympatric rock-dwelling cichlids of Lake Tanganyika, Eretmodus cyanostictus, Tropheus moorii, and Ophthalmotilapia ventralis. The species represent three endemic, phylogenetically distinct tribes (Eretmodini, Tropheini, and Ectodini), and display divergent ecomorphological and behavioral specialization. Sample locations span both continuous, rocky shoreline and a potential dispersal barrier in the form of a muddy bay. High genetic diversity and population differentiation were detected in T. moorii and E. cyanostictus, whereas much lower variation and structure were found in O. ventralis. In particular, while a 7-km-wide muddy bay curtails dispersal in all three species to a similar extent, gene flow along mostly continuous habitat appeared to be controlled by distance in E. cyanostictus, further restricted by site philopatry and/or minor habitat discontinuities in T. moorii, and unrestrained in O. ventralis. In contrast to the general pattern of high gene flow along continuous shorelines in rock-dwelling cichlids of Lake Malawi, our study identifies differences in population structure among stenotopic Lake Tanganyika species. The amount of genetic differentiation among populations was not related to the degree of geographical variation of body color, especially since more phenotypic variation is observed in O. ventralis than in the genetically highly structured E. cyanostictus.

Phylogeography and evolution of the Tanganyikan cichlid genus Tropheus based upon mitochondrial DNA sequences.

Lake Tanganyika harbors the oldest and most diverse species flock of cichlid fish. Many species are subdivided into numerous genetically and phenotypically distinct populations. Their present distribution and genetic structure were shaped by a series of lake level fluctuations which caused cycles of isolation and admixis and promoted dispersal events. One of the best examples of this phenomenon is the genus Tropheus. We present a comprehensive mtDNA phylogeny based upon 365 individuals of 55 populations from all over Lake Tanganyika, which suggests an almost-contemporaneous origin of eight major mitochondrial lineages about 700 Ka ago. While the distribution of seven lineages is restricted to particular sections of the lake shore, one lineage was found to have a much more widespread distribution. This particular lineage is subdivided into four sublineages which seem to have originated from a single dispersal event about 400 Ka. By using a molecular clock estimate in combination with geological data we derived a hypothetical scenario for the colonization history of Tropheus. Thereby we show a high degree of concordance between major changes of the lake level in the recent history of Lake Tanganyika and three distinct diversification events in this genus.

Phylogeny of the Lake Tanganyika cichlid species flock and its relationship to the Central and East African haplochromine cichlid fish faunas.

Lake Tanganyika, the oldest of the East African Great Lakes, harbors the ecologically, morphologically, and behaviorally most complex of all assemblages of cichlid fishes, consisting of about 200 described species. The evolutionary old age of the cichlid assemblage, its extreme degree of morphological differentiation, the lack of species with intermediate morphologies, and the rapidity of lineage formation have made evolutionary reconstruction difficult. The number and origin of seeding lineages, particularly the possible contribution of riverine haplochromine cichlids to endemic lacustrine lineages, remains unclear. Our phylogenetic analyses, based on mitochondrial DNA sequences of three gene segments of 49 species (25% of all described species, up to 2,400 bp each), yield robust phylogenies that provide new insights into the Lake Tanganyika adaptive radiation as well as into the origin of the Central- and East-African haplochromine faunas. Our data suggest that eight ancient African lineages may have seeded the Tanganyikan cichlid radiation. One of these seeding lineages, probably comprising substrate spawning Lamprologus-like species, diversified into six lineages that evolved mouthbrooding during the initial stage of the radiation. All analyzed haplochromines from surrounding rivers and lakes seem to have evolved within the radiating Tanganyikan lineages. Thus, our findings contradict the current hypothesis that ancestral riverine haplochromines colonized Lake Tanganyika to give rise to at least part of its spectacular endemic cichlid species assemblage. Instead, the early phases of the Tanganyikan radiation affected Central and East African rivers and lakes. The haplochromines may have evolved in the Tanganyikan basin before the lake became a hydrologically and ecologically closed system and then secondarily colonized surrounding rivers. Apparently, therefore, the current diversity of Central and East African haplochromines represents a relatively young and polyphyletic fauna that evolved from or in parallel to lineages now endemic to Lake Tanganyika.