Disease Of Stone Fruits Research Articles

Deciphering the Monilinia fructicola Genome to Discover Effector Genes Possibly Involved in Virulence.

Brown rot is the most economically important fungal disease of stone fruits and is primarily caused by Monilinia laxa and Monlinia fructicola. Both species co-occur in European orchards although M. fructicola is considered to cause the most severe yield losses in stone fruit. This study aimed to generate a high-quality genome of M. fructicola and to exploit it to identify genes that may contribute to pathogen virulence. PacBio sequencing technology was used to assemble the genome of M. fructicola. Manual structural curation of gene models, supported by RNA-Seq, and functional annotation of the proteome yielded 10,086 trustworthy gene models. The genome was examined for the presence of genes that encode secreted proteins and more specifically effector proteins. A set of 134 putative effectors was defined. Several effector genes were cloned into Agrobacterium tumefaciens for transient expression in Nicotiana benthamiana plants, and some of them triggered necrotic lesions. Studying effectors and their biological properties will help to better understand the interaction between M. fructicola and its stone fruit host plants.

Considerations on the management and prevention of virus and virus-like diseases of stone fruits in Uruguay

The following agents were reported in Uruguay in stone fruit trees: PNRSV (Prunus necrotic ringspot virus), PDV (Prune dwarf virus), ACLSV (Apple chlorotic leafspot virus), ApMV (Apple mosaic virus) and PLMVd (Peach latent mosaic viroid). PNRSV and PDV are widespread, their plant to plant transmission in the orchard and by seed was confirmed and causes important losses in nurseries and commercial plantations. ACLSV, ApMV y PLMVd are not widely disseminated, and PPV (causal agent of sharka, Plum pox virus) was not found. Certification of fruit tree plants supervised by the INASE is mandatory. Healthy propagative materials of cultivars and rootstocks are available, but growers prefer the cheapest plants with less coverage of certification. A limited, uneven, and decreasing demand for healthy plants threatens the sustainability of the system. Phytosanitary requirements to prevent the entry of new pests (quarantine) are established by Ministry of Livestock, Agriculture and Fisheries. Official phytosanitary documents required are often difficult to obtain by the suppliers of new cultivars or rootstocks and the risk of pest introduction is high. For this reason, many countries have post entry quarantine stations where introduced plant materials are kept under surveillance until release to the industry. Some fruit producing regions that have important diseases perform plant eradication programs, control of vectors and replacement with certified plants; but, according to the current sanitary status of stone fruit trees in Uruguay, that would not be necessary yet. Conversely, it would be advisable to: 1) prevent the arrival of new pests, considering to set post quarantine plant stations, 2) keep an active surveillance to early detect sanitary problems, 3) encourage sanitary quality on certified plants and 4) develop new orchards using virus-free certified plants.

Screening of germplasm of peach and nectarines against Taphrina deformans

The leaf curl caused by Taphrina deformans (Berk.) Tul. is the most serious disease of stone fruits viz., peachs and nectarines which counts for loss in quantitative and qualitative yield. Germplasm of these stone fruits was screened against T. deformans and data in terms of disease incidence (%) and disease severity (%) were recorded. It was observed that in peach, cent percent disease incidence was recorded in Glo haven, Pratap, Shan-e-punjab and Florida Peach while, minimum in Androse (30%) whereas, maximum disease severity was observed in Red Haven (1.13%), Pratap (1.10%), Early Elberta (1.07%) and minimum in Early Grande (0.53%). In nectarine, cent per cent disease incidence was observed in Independence, Early Red Haven, Punjab Nectarine and Syria while, minimum disease incidence (33.33%) and severity (0.3%) was recorded in Silver of Rome whereas, maximum disease severity was observed in Red Gold (1.58%), Independence (1.2%) and Early Red Haven (1.2%).

Field validation of biocontrol strategies to control brown rot on stone fruit in several European countries.

Brown rot caused by Monilinia spp. is the most significant disease of stone fruit. New approaches to fruit production have necessitated the development of control strategies that are more eco- and consumer-friendly. An efficient field strategy to control brown rot was previously designed based on the application of two biocontrol agents (BCAs), Bacillus amyloliquefaciens CPA-8 (CPA-8) or Penicillium frequentans 909 (Pf909), with calendar-based treatment. In the present study, the strategy was validated on different stone fruit hosts in four producing countries over two seasons. The results obtained were reported according to three different scenarios: Scenario 1, in which there was no presence of disease in the field; Scenario 2, in which high disease pressure occurred in the field and treatments (biologicals or chemicals) were not effective; and Scenario 3, with low or medium to high disease presence. The results were successful because, in general, BCA strategies were shown to control brown rot to a similar extent as chemicals strategies. We found that most of the trials conducted in this study were classed under Scenario 3 (62.5%), with only 12.5% and 25% of the trials classed under Scenarios 1 and 2, respectively. These novel findings allowed the formulation of CPA-8 and Pf909 as valuable tools for farmers to produce stone fruits more competitively and meet consumer demand for safer and more environmentally friendly products. © 2021 Society of Chemical Industry.

Studies of Resistance to Sharka (Plum Pox) in Romanian Apricot Progenies

Sharka (Plum pox) is considered one of the most devastating diseases of stone fruits in terms of agronomic impact and economic importance. The response of several apricot hybrids was evaluated over the vegetative period of 2010 by visual monitoring of symptom development and by serological and molecular methods. After two cycles of study, all the replicates of ‘Mari de Cenad’, ‘Traian’ and ‘Tabriz’ showed sharka symptoms while the replicates of ‘Stark Early Orange’ and NJA 2 did not show any symptoms and were not ELISA-positive. The resistant progenitors (‘Stark Early Orange’, NJA17, NJA42 and NJA2) were able to transmit PPV resistance to their descendants, in agreement with previous results (DOSBA et al, 1994, MARTINEZ-GOMEZ et al, 2000; AUDERGON et al, 1994). The genetic control hypotheses for PPV resistance in apricot referenced by different authors considered the resistance allele as dominant. Romanian apricot F1 and F2 progenies evaluated were classified into two groups: susceptible to PPV and resistant to PPV. After that the most important genotypes were grafted onto infected GF 305 (considered as indicator for PPV) in greenhouse conditions. The final step is the introduction and development of the marker-assisted selection (MAS) in PPV resistance F1 and F2 progenies in apricot Romanian genotypes. DNA was isolated from all hybrids and parent genotypes. These samples will be used to implement the molecular markers for identification of genes linked to plant response to PPV infection.

Genotypic differentiation of Monilinia spp. populations in Serbia using a high-resolution melting (HRM) analysis

Monilinia laxa, Monilinia fructicola and Monilinia fructigena are the three main causal agents of brown rot, which is one of the most important diseases of stone fruits in pre- and postharvest conditions. Nowadays, the need for the precise genotyping of these Monilinia species in terms of the genetic diversity of their populations or differences in their pathogenicity and host range is a prerequisite for any efficient disease management. In our study, the genetic structure of Monilinia populations in Serbia from three geographically distinct regions was investigated employing <br /> a high-resolution melting (HRM) analysis which is a sensitive and rapid molecular approach in fungal ge­notyping and diagnostics. Using species-specific primer pairs genotype-specific HRM melting curve profiles were generated allowing to efficiently decipher the genetic diversity of the Monilinia populations. The Monilinia genotypes could be easily distinguished according to their melting curves. The isolates from the northern region were assigned to distinct genotypes and grouped rather independently compared to the isolates of the other two regions among all three tested Monilinia spp. M. fructicola and M. fructigena showed a higher genetic diversity among their populations (44%) compared with the genetic diversity among the M. laxa populations (7%). In contrast, the genetic variance within the pathogen populations was higher in the case of M. laxa (93%). Our data revealed an absence of host specificity in the Monilinia spp. populations.

First report of blue mold caused by Penicillium crustosum on nectarine fruit in Serbia.

Penicillium crustosum Thom. is a fungus commonly found on cheese and nuts, but is also a postharvest pathogen that causes blue mold disease of pome and stone fruits including plum and nectarine (Louw and Korsten 2016; Restuccia et al. 2006). The fungus produces mycotoxins (penitrem A, roquefortine C, terrestric acid, and cyclopenol) which are of concern for human health (Frisvad and Samson 2004). In Serbia, P. crustosum has been previously described on apple fruit (Vico et al. 2014). On nectarine fruit (Prunus persica var. nucipersica), after 6 weeks of cold storage, symptoms of blue mold developed in a fruit market in Belgrade, Serbia. The fruit was collected and isolations performed in November 2017. Decayed areas on infected fruit were soft, light to medium brown with blue-green sporulation on the fruit surface. Two isolates were obtained (N2AS and N2BS) and cultured on Czapek yeast autolysate agar (CYA), malt extract agar (MEA), yeast extract sucrose agar (YES) and potato dextrose agar (PDA) at 25°C for 7 days. Isolates were identified as P. crustosum based on morphological features (Frisvad and Samson 2004; Pitt and Hocking 2009). On all media, mycelia were white and colonies turned blue-green with abundant sporulation. Colonies of both isolates were radially sulcate on MEA and YES, and plane with a granular texture on CYA and PDA, and were yellow to orange on the reverse side on YES. Mean colony diameter on PDA was 29.2 ± 1.2 mm for N2AS, and 31.3 ± 1.4 mm for N2BS; on CYA 30.8 ± 1.2 mm for N2AS and 30.9 ± 1.1 mm for N2BS; on YES 40.7 ± 3.6 mm for N2AS and 43.6 ± 1.4 mm for N2BS; and on MEA 33.4 ± 1.2 mm for N2AS and 34 ± 2.5 mm for N2BS. Crusts of conidial masses formed on MEA and PDA after 10 days. Conidiophores of both isolates were terverticillate, stipes were septate with rough walls, and conidia, borne in columns, were smooth and spherical to subglobose. Conidial diameter for N2AS was 2.32 to 3.95 (average 3.13) µm and for N2BS was 2.34 to 3.98 (average 3.27) µm (n=50). Isolates formed a yellow ring, using Ehrlich's reagent, indicating lack of cyclopiazonic acid, but production of other alkaloids. Morphological identification was confirmed by isolating genomic DNA, PCR amplification of the partial β-tubulin gene using Bt2a/Bt2b (Glass and Donaldson 1995) and sequencing. BLAST analysis revealed that N2AS sequence (MT799805) was 99% similar and N2BS (MT799806) was identical to sequences AY674351 (strain CBS 101025) and KJ775121 (strain DTO_244H8) of P. crustosum in GenBank. Sequences (2X consensus) of the two isolates differed in one nucleotide showing the existence of single-nucleotide polymorphism among P. crustosum isolates. Pathogenicity was tested on nectarine, peach and apple fruit (four fruit per isolate and the control). Fruit were washed, surface-sanitized with 70% ethanol, and wound (10x4 mm) inoculated on two sides with 40 μl of a 105/ml conidial suspension in sterile distilled water containing 0.1% Tween 20 (TSDW). Control fruit was inoculated with TSDW. Inoculated and control fruit were stored at 25°C for 7 days. Inoculated fruit developed light brown decay with cracks in the epidermis that spread from the inoculation point on nectarines and peaches. Blue-green sporulation was present on all inoculated fruit. Control fruit remained symptomless. The fungus was re-isolated and was morphologically identical to the original isolates, thus completing Koch's postulates. This is the first report of P. crustosum causing postharvest blue mold decay on nectarine fruit in Serbia. Results show that P. crustosum is not only present as a postharvest pathogen of apple fruit, but of nectarine as well and may pose a threat in storage of both pome and stone fruits in Serbia.

Grey mould populations in northern German sweet cherry and plum orchards: selection of fungicide-resistant Botrytis cinerea strains over sensitive B. pseudocinerea by fungicide treatments

Second only to brown rot caused by Monilinia spp., grey mould caused by Botrytis spp. is one of the most important pre- and post-harvest diseases of stone fruit in northern Germany. Among 484 Botrytis isolates from sweet cherries produced under integrated pest management (IPM), 60.3% showed resistance to QoI fungicides and 54.6% to boscalid, reflecting the heavy use of these compounds in regional production. More modest resistance frequencies were recorded for fenhexamid (20.7%), fluopyram (8.3%), fludioxonil (9.1%) and cyprodinil (21.9%). A 7.6% share of isolates possessed multiple resistance to at least five of these fungicides. Resistance was also detected for two fungicides no longer registered in cherries, viz. thiophanate-methyl (15.7%) and iprodione (20.9%). Similar results were obtained for 141 Botrytis isolates from IPM plum orchards. In contrast, in organically produced cherries (88 Botrytis isolates) resistance frequencies were < 20% for all fungicides. The dominant species on cherries and plums was B. cinerea. The share of B. pseudocinerea, which is not known to have developed resistance to commercial fungicides, was 18.0% in cherries and 9.9% in plums from IPM, but 50.0% in organically produced cherries. A 10-year annual survey of a sweet cherry orchard under IPM showed the immigration of multi-resistant B. cinerea strains in 2011, followed by their increase and decline in subsequent years. An understanding of the effects of fungicide spray regimes on Botrytis population dynamics in stone fruit has implications for resistance management. In practice, a limitation of the number of pre-harvest sprays to a maximum of two will give a satisfactory control of brown rot in most situations, while a higher number will promote multi-resistant B. cinerea strains.

Morphology, DNA Phylogeny, and Pathogenicity of Wilsonomyces carpophilus Isolate Causing Shot-Hole Disease of Prunus divaricata and Prunus armeniaca in Wild-Fruit Forest of Western Tianshan Mountains, China

Prunus divaricata and Prunus armeniaca are important wild fruit trees that grow in part of the Western Tianshan Mountains in Central Asia, and they have been listed as endangered species in China. Shot-hole disease of stone fruits has become a major threat in the wild-fruit forest of the Western Tianshan Mountains. Twenty-five isolates were selected from diseased P. divaricata and P. armeniaca. According to the morphological characteristics of the culture, the 25 isolates were divided into eight morphological groups. Conidia were spindle-shaped, with ovate apical cells and truncated basal cells, with the majority of conidia comprising 3–4 septa, and the conidia had the same shape and color in morphological groups. Based on morphological and cultural characteristics and multilocus analysis using the internal transcribed spacer (ITS) region, partial large subunit (LSU) nuclear ribosomal RNA (nrRNA) gene, and the translation elongation factor 1-alpha (tef1) gene, the fungus was identified as Wilsonomyces carpophilus. The 25 W. carpophilus isolates had high genetic diversity in phylogenetic analysis, and the morphological groups did not correspond to phylogenetic groups. The pathogenicity of all W. carpophilus isolates was confirmed by inoculating healthy P. divaricata and P. armeniaca leaves and fruits. The pathogen was re-isolated from all inoculated tissues, thereby fulfilling Koch’s postulates. There were no significant differences in the pathogenicity of different isolates inoculated on P. armeniaca and P. divaricata leaves (p &gt; 0.05). On fruit, G053 7m3 and G052 5m2 showed significant differences in inoculation on P. armeniaca, and G010 5m2 showed extremely significant differences with G004 7m2 and G004 5m2 on P. divaricata (p &lt; 0.05). This is the first report on shot-hole disease of P. armeniaca (wild apricot) leaves and P. divaricata induced by W. carpophilus in China.

Thyrostroma carpophilum insertional mutagenesis: A step towards understanding its pathogenicity mechanism

Thyrostroma carpophilum, a causal agent of shot hole disease of stone fruits, cause severe loss in economically important fruit crops of Kashmir. Understanding its pathogenesis at molecular level will aid in devising a better management strategy. In this study, we optimized Agrobacterium tumefaciens mediated transformation (ATMT) conditions for T. carpophilum using PBIF2-EGFP construct. Using this protocol, we obtained 328 positive transformants per 104 spores and subsequent sub-culturing of transformants on selective and non-selective media resulted in stable T-DNA integration. Southern blot analysis revealed that most of the transformants embodied single T-DNA integration. Using this method, we obtained a small-scale transformant library (2050 transformants). Among this pool, we tested 1005 transformants for their pathogenicity; out of which 185 showed complete pathogenicity loss, 35 displayed reduced virulence and 785 were pathogenically similar to wild type. Out of this experimental stock, three transformants from each category were randomly selected to dissect the infection assay. The findings deciphered that transformants with complete pathogenicity loss failed to penetrate the host tissue and a few transformants failed to sporulate in laboratory. Transformants from reduced category could not form appressorium and occasionally sporulated. Transformants similar to wild type were morphologically and pathogenically similar to wild type because of un-alteration in their modus operandi. Our work provides a new platform to understand the pathogenicity mechanism of T. carpophilum. The optimized ATMT protocol will help in developing large transformant library that can help to identify the virulence arsenals necessary for the pathogen to cause disease.

Occurrence of Xanthomonas arboricola pv. pruni Causing Bacterial Leaf Spot and Shot-Hole on Peach in Montenegro

During May and June 2019, bacterial leaf spot was observed on peach (Prunus persica L. Batsch) in a young plantation (2017, 2018) on four different cultivars (Sugar Time, Britney Lane, Royal Bell, and Royal Time) in the Podgorica region of Montenegro. Diseased trees appeared sporadically, and incidence of symptomatic leaves per infected tree was 10 to 15%. Angular water-soaked spots surrounded by chlorotic tissue were observed along leaf midribs or margins, turning light brown in later stages. Necrotic lesions sometimes dropped out, giving the leaves a “shot-hole” appearance. When spots coalesced, large areas of necrotic foliar tissue formed. Eventually leaves turned yellow, resulting in premature defoliation. Margins taken from healthy tissue and necrotic lesions were homogenized in sterile distilled water (SDW) and spread onto YDC agar (Schaad et al. 2001), on which mostly pale yellow, translucent, circular, raised, and mucoid Xanthomonas-like colonies were observed after 3 days incubation at 26°C. Twelve representative isolates coded as Xp1 to 12 (Sugar Time, Xp1 to 3; Britney Lane, Xp4 to 6; Royal Bell, Xp7 to 9; and Royal Time, Xp10 to 12) were aerobic, gram negative, catalase positive, and oxidase and arginine dihydrolase negative; hydrolyzed esculin and gelatin but not starch; produced H₂S but not indole; and did not reduce nitrate (Schaad et al. 2001). All reactions corresponded with the reactions of the reference strain NCPPB 3156 of Xanthomonas arboricola pv. pruni (Xap), causal agent of leaf spot, shot-hole, and canker of stone fruit. The identity of the isolates was confirmed by PCR using the Xap-specific primer pair XapY17-F/XapY17-R for the ABC transporter ATP-binding protein (Pothier et al. 2011). A single unique target band of 943 bp was amplified for all isolates tested. Furthermore, sequencing of the housekeeping genes fyuA, rpoD, and gyrB (Young et al. 2008) showed 100% identity of tested isolates with genomic sequences of Xap strains deposited in the NCBI database (CFBP 411, 3921, 5580, 6653, 7099, and 7100) based on BLAST analysis and a neighbor-joining phylogenetic tree. The sequences of four isolates representing each cultivar were deposited in GenBank under accession numbers MN520623 to 26 for fyuA, MN520627 to 30 for rpoD, and MN520631 to 34 for gyrB gene for Xp1, Xp4, Xp7, and Xp10, respectively. Pathogenicity was confirmed on (i) young, fully expanded, healthy, detached peach leaves using a hypodermic syringe without a needle, pressed against the abaxial side of the leaf (bacterial suspension at 10⁷ CFU/ml from a 72-h YDC culture) and (ii) immature fruits stabbed and pressing the bacterial suspension (10⁷ CFU/ml) until overflow. Xap reference strain NCPPB 3156 served as a positive and SDW as a negative control. Inoculated leaves were kept under controlled conditions at 25°C, 16/8-h day/night photoperiod, and 60 to 80% relative humidity. All tested bacterial isolates and the reference strain developed water-soaked spots on leaves 3 days and on fruits 4 days after inoculation. Ten days after inoculation the spots became dark brown and necrotic. SDW-treated controls were negative. Xap was successfully reisolated and confirmed to be Xap using PCR (XapY17-F/XapY17-R). This study demonstrates Xap on peach as a new host in Montenegro. Xap was previously reported from other (15 of 44) European countries (https://gd.eppo.int/taxon/XANTPR/distribution), mainly on peach and plum, and from Montenegro on almond (Panic et al. 1998). As the causal agent of a major disease of stone fruits, Xap is a quarantine organism in Europe (EPPO A2 List of Pests recommended for regulation for EU). Therefore, it is necessary to implement containment and/or eradication measures to prevent further spread to new areas and/or new hosts. Furthermore, a nation-wide survey on the (re)occurrence of Xap in other hosts such as almond (reported by Panic et al. 1998), apricot, and plum advised.

Translatome Profiling of Plum Pox Virus-Infected Leaves in European Plum Reveals Temporal and Spatial Coordination of Defense Responses in Phloem Tissues.

Plum pox virus (PPV) is the causative agent of sharka, a devastating disease of stone fruits including peaches, apricots, and plums. PPV infection levels and associated disease symptoms can vary greatly, depending upon the virus strain, host species, or cultivar as well as developmental age of the infected tissues. For example, peaches often exhibit mild symptoms in leaves and fruit while European plums typically display severe chlorotic rings. Systemic virus spread into all host tissues occurs via the phloem, a process that is poorly understood in perennial plant species that undergo a period of dormancy and must annually renew phloem tissues. Currently, little is known about how phloem tissues respond to virus infection. Here, we used translating ribosome affinity purification followed by RNA sequencing to identify phloem- and nonphloem-specific gene responses to PPV infection during leaf development in European plum (Prunus domestica L.). Results showed that, during secondary leaf morphogenesis (4- and 6-week-old leaves), the phloem had a disproportionate response to PPV infection with two- to sixfold more differentially expressed genes (DEGs) in phloem than nonphloem tissues, despite similar levels of viral transcripts. In contrast, in mature 12-week-old leaves, virus transcript levels dropped significantly in phloem tissues but not in nonphloem tissues. This drop in virus transcripts correlated with an 18-fold drop in phloem-specific DEGs. Furthermore, genes associated with defense responses including RNA silencing were spatially coordinated in response to PPV accumulation and were specifically induced in phloem tissues at 4 to 6 weeks. Combined, these findings highlight the temporal and spatial dynamics of leaf tissue responses to virus infection and reveal the importance of phloem responses within a perennial host.

New High-Quality Draft Genome of the Brown Rot Fungal Pathogen Monilinia fructicola.

Brown rot is a worldwide fungal disease of stone and pome fruit that is caused by several Monilinia species. Among these, Monilinia fructicola can cause severe preharvest and postharvest losses, especially for stone fruit. Here, we present a high-quality draft genome assembly of M. fructicola Mfrc123 strain obtained using both Illumina and PacBio sequencing technologies. The genome assembly comprised 20 scaffolds, including 29 telomere sequences at both ends of 10 scaffolds, and at a single end of 9 scaffolds. The total length was 44.05 Mb, with a scaffold N50 of 2,592 kb. Annotation of the M. fructicola assembly identified a total of 12,118 genes and 13,749 proteins that were functionally annotated. This newly generated reference genome is expected to significantly contribute to comparative analysis of genome biology and evolution within Monilinia species.

Exploring sources of resistance to brown rot in an interspecific almond × peach population.

Monilinia spp. are responsible for brown rot, one of the most significant stone fruit diseases. Planting resistant cultivars seems a promising alternative, although most commercial cultivars are susceptible to brown rot. The aim of this study was to explore resistance to Monilinia fructicola over two seasons in a backcross one interspecific population between almond 'Texas' and peach 'Earlygold' (named T1E). 'Texas' almond was resistant to brown rot inoculation, whereas peach was highly susceptible. Phenotypic data from the T1E population indicated wide differences in response to M. fructicola. Additionally, several non-wounded individuals exhibited resistance to brown rot. Quantitative trait loci (QTLs) were identified in several linkage groups, but only two proximal QTLs in G4 were detected over both seasons and accounted for 11.3-16.2% of the phenotypic variation. Analysis of the progeny allowed the identification of resistant genotypes that could serve as a source of resistance in peach breeding programs. The finding of loci associated with brown rot resistance would shed light on implementing a strategy based on marker-assisted selection (MAS) for introgression of this trait into elite peach materials. New peach cultivars resistant to brown rot may contribute to the implementation of more sustainable crop protection strategies. © 2019 Society of Chemical Industry.

Integration of biological and chemical control of brown rot of stone fruits to reduce disease incidence on fruits and minimize fungicide residues in juice

Brown rot is a major disease of stone fruits caused by Monilinia spp. It can reach high incidence that leads to considerable losses during storage. Our work aimed at the evaluation of innovative biological and integrated approaches to control brown rot of apricots and peaches, and of their influence on the presence of fungicide residues both in the harvested fruits and derived peach juices. In this work, we report the results of a two-year field investigation carried out in 5 different farms in Southern Italy. Plant health management strategy was based on two treatments performed in the last thirty days before harvest. Two biocontrol agents (BCAs), the yeast Rhodotorula kratochvilovae strain LS11 and the commercial biofungicide Serenade Max® (based on Bacillus subtilis strain QST 713), were applied alone (biocontrol) or in combination with low doses of the fungicides (25% of full dosage, integrated control) cyprodinil (30 days before harvest), and boscalid (7 days before harvest). The highest protection was recorded in conventional (fungicides applied at full dosage) and integrated control treatments, both on apricots and peaches. In the integrated control, residues of fungicides in fruits and in peach juice were significantly lower compared to those of the conventional control in both fruit species. Our data show that biological and integrated control based on two key treatments at thirty and seven days before harvest can be considered as effective and safer control strategies of brown rot of stone fruits. Furthermore, the applied strategies can dramatically reduce and even set to zero the level of fungicide residues in derived fruit juices.

Improving profitability: assessment of new rootstocks and planting systems

For over 25 years, rootstocks for sweet cherries are evaluated in different locations in the german federal state of Rhenania Palatinate. They are situated in a vine growing climate and have medium-heavy soils. Replant disease of stone fruit plays a significant role in most orchards. In general, all trees in the rootstock trials are trained as central leader and planted 4.0 to 4.5 m by 2.0 to 3.0 m depending on the expected vigour. In addition to the common performance parameters, tree health, anchorage and tendency for root suckers are observed. In 2013 a new rootstock trial has been established including, inter alia, the new Weigi series and some recent Gisela numbers. In the experimental orchard of Oppenheim, replanted soil can be compared to new land. Up to now, Weigi 2 is slightly more dwarfing but also less productive in this trial what is no disadvantage regarding the fruit size. In the group of semi-dwarfing rootstocks Weigi 1, Weigi 3, Gisela 13 and Gisela 17 seem to be similar both in vigour and yield efficiency. In this group there is an interest to replace PiKu 1 which turned out to be not winter hardy enough and susceptible to heavy soils and bark beetles under German conditions. Training trials revealed that also with low tree forms, yields and qualities comparable to the spindle can be obtained. The picking performance can be improved in many cases. Mechanical pruning only makes sense if it is combined with manual correction pruning. An exclusive mechanical pruning reduces the fruit size and can also reduce the picking performance.

Monilinia fructicola and Monilinia laxa isolates from stone fruit orchards sprayed with fungicides displayed a broader range of responses to fungicides than those from unsprayed orchards

Monilinia fructicola and Monilinia laxa are causal agents of brown rot, the most serious fungal disease of stone fruit (Prunus species). The disease is controlled primarily by applying fungicides. It was hypothesised that Monilinia isolates exposed to a regime of fungicidal sprays would exhibit greater tolerance to those compounds than isolates that had not been subjected at all to such fungicide sprays. Sixty-six M. fructicola and 52 M. laxa isolates were collected from fungicide-sprayed and unsprayed commercial and domestic orchards. The fungicides propiconazole, iprodione, and a mixture of fluopyram and trifloxystrobin were used regularly on all the sprayed orchards tested, and these were used to challenge all Monilinia isolates in vitro. We found that isolates collected from sprayed orchards were on average more tolerant to the fungicides, as measured by effective concentration of fungicide reducing mycelial growth by 50% (EC50). This trend was evident for both fungal species tested, but it was statistically significant only for M. fructicola. Monilinia laxa isolates were on average more tolerant to propiconazole than were M. fructicola isolates irrespective of orchard type, while average responses to iprodione and fluopyram + trifloxystrobin were similar for both species. Although tolerant and sensitive isolates were identified under both sprayed and unsprayed regimes, there was a greater range of responses to all three fungicides by isolates from sprayed orchards. Isolates with tolerance to two fungicides were not exclusively from sprayed orchards, but occurred more frequently there.

Mating System in the Brown Rot Pathogens Monilinia fructicola, M. laxa, and M. fructigena.

Monilinia fructicola, M. laxa, and M. fructigena are the most important pathogens responsible for brown rot disease of stone and pome fruits. Information on their mating system and sexual behavior is scant. A mating-type-specific PCR-based assay was developed and applied to 155 Monilinia isolates from 10 countries and 10 different host plants. We showed that single isolates carry only one of two opposite idiomorphs at the MAT1 locus consistent with a heterothallic mating system for all three species. MAT1-1 and MAT1-2 mating types were detected in similar proportions in samples of isolates of each species and hence there do not appear to be genetic obstacles to the occurrence of sexual reproduction in their populations. Inter simple sequence repeat markers suggested that asexual reproduction is prevalent, but that sexual recombination occurs in M. fructicola populations in Italy. The genetic architectures of the MAT1 loci of the three pathogens were analyzed. MAT1-1 and MAT1-2 idiomorphs are flanked upstream and downstream by the APN2 and SLA2 genes and resemble those of Botrytis cinerea and other heterothallic fungi in the family Sclerotiniaceae. Each idiomorph contains a specific couple of genes, MAT1-1-1 (with alpha-box domain) and MAT1-1-5 in MAT1-1, and MAT1-2-1 (with HMG-box domain) and MAT1-2-10 in MAT1-2. Small gene fragments (dMAT1-1-1 and dMAT1-2-1) from the opposite idiomorph were detected close to their flanking regions. Constitutive expression of the four MAT1 genes during vegetative growth was ascertained by transcriptomic analysis (RNA-Seq). Antisense transcription of the MAT1-1-1 and MAT1-2-1 genes and intergenic transcribed regions of the MAT1 locus were detected. These results represent new insights into the mating systems of these three economically-important pathogens which could contribute to improve the knowledge on their population biology.

Draft Genome Resources for the Phytopathogenic Fungi Monilinia fructicola, M. fructigena, M. polystroma, and M. laxa, the Causal Agents of Brown Rot.

Fungi in the genus Monilinia cause brown rot disease of stone and pome fruits. Here, we report the draft genome assemblies of four important phytopathogenic species: M. fructicola, M. fructigena, M. polystroma, and M. laxa. The draft genome assemblies were 39 Mb (M. fructigena), 42 Mb (M. laxa), 43 Mb (M. fructicola), and 45 Mb (M. polystroma) with as few as 550 contigs (M. laxa). These are the first draft genome resources publicly available for M. laxa, M. fructigena, and M. polystroma.

De novo assembly and comparative transcriptome analysis of Monilinia fructicola, Monilinia laxa and Monilinia fructigena, the causal agents of brown rot on stone fruits

BackgroundBrown rots are important fungal diseases of stone and pome fruits. They are caused by several Monilinia species but M. fructicola, M. laxa and M. fructigena are the most common all over the world. Although they have been intensively studied, the availability of genomic and transcriptomic data in public databases is still scant. We sequenced, assembled and annotated the transcriptomes of the three pathogens using mRNA from germinating conidia and actively growing mycelia of two isolates of opposite mating types per each species for comparative transcriptome analyses.ResultsIllumina sequencing was used to generate about 70 million of paired-end reads per species, that were de novo assembled in 33,861 contigs for M. fructicola, 31,103 for M. laxa and 28,890 for M. fructigena. Approximately, 50% of the assembled contigs had significant hits when blasted against the NCBI non-redundant protein database and top-hits results were represented by Botrytis cinerea, Sclerotinia sclerotiorum and Sclerotinia borealis proteins. More than 90% of the obtained sequences were complete, the percentage of duplications was always less than 14% and fragmented and missing transcripts less than 5%. Orthologous transcripts were identified by tBLASTn analysis using the B. cinerea proteome as reference. Comparative transcriptome analyses revealed 65 transcripts over-expressed (FC ≥ 8 and FDR ≤ 0.05) or unique in M. fructicola, 30 in M. laxa and 31 in M. fructigena. Transcripts were involved in processes affecting fungal development, diversity and host-pathogen interactions, such as plant cell wall-degrading and detoxifying enzymes, zinc finger transcription factors, MFS transporters, cell surface proteins, key enzymes in biosynthesis and metabolism of antibiotics and toxins, and transposable elements.ConclusionsThis is the first large-scale reconstruction and annotation of the complete transcriptomes of M. fructicola, M. laxa and M. fructigena and the first comparative transcriptome analysis among the three pathogens revealing differentially expressed genes with potential important roles in metabolic and physiological processes related to fungal morphogenesis and development, diversity and pathogenesis which need further investigations. We believe that the data obtained represent a cornerstone for research aimed at improving knowledge on the population biology, physiology and plant-pathogen interactions of these important phytopathogenic fungi.