Identification of novel genetic regions associated with resistance to European canker in apple

Resistance to Neonectria ditissima, the fungus causing European canker in apple, was studied in a multiparental population of apple scions using several phenotyping methods. The studied population consists of individuals from multiple families connected through a common pedigree. The degree of disease of each individual in the population was assessed in three experiments: artificial inoculations of detached dormant shoots, potted trees in a glasshouse and in a replicated field experiment. The genetic basis of the differences in disease was studied using a pedigree-based analysis (PBA). Three quantitative trait loci (QTL), on linkage groups (LG) 6, 8 and 10 were identified in more than one of the phenotyping strategies. An additional four QTL, on LG 2, 5, 15 and 16 were only identified in the field experiment. The QTL on LG2 and 16 were further validated in a biparental population. QTL effect sizes were small to moderate with 4.3 to 19 % of variance explained by a single QTL. A subsequent analysis of QTL haplotypes revealed a dynamic response to this disease, in which the estimated effect of a haplotype varied over the field time-points. Two groups of QTL-haplotypes could be distinguished, one that displayed increased effect and one with a constant effect across time-points. These results suggest that there are different modes of control of N. ditissima in the early stages of infection compared to later time-points of disease development. It also shows that multiple QTL will need to be considered to improve resistance to European canker in apple breeding germplasm.

Response of different grapevine cultivars to infection by Lasiodiplodia theobromae and Lasiodiplodia mediterranea

Botryosphaeria dieback is a grapevine trunk disease that affects all viticulture regions of the world. Species of the genus Lasiodiplodia have been reported as pathogenic towards grapevine in several growing regions and have also been previously reported from Portuguese vineyards. Species in this genus, particularly Lasiodiplodia theobromae, have been reported on previous studies to be more aggressive than other Botryosphaeriaceae species most commonly associated with Botryosphaeria dieback. The aim of this study was to assess the response of some of the more representative cultivars planted throughout Portuguese vineyards, Touriga Nacional, Touriga Franca, Alvarinho, Aragonez (=Tempranillo) and Cabernet Sauvignon, by performing artificial inoculations with Lasiodiplodia spp. collected in different geographic locations worldwide. Two experiments, one by inoculating two-year-old grapevines kept on a greenhouse-controlled conditions with six isolates of L. theobromae and one isolate of L. mediterranea and other by inoculating seven-year-old field grown grapevines with two isolates of L. theobromae, were conducted twice. Response of the cultivars was assessed by evaluating the lesion length caused by the isolates under study, five months after inoculation. The results showed that all isolates studied were able to infect the annual shoots since they were always re-isolated and produced internal wood discoloration. Significant differences were found for all isolate/cultivar combinations. For both experiments, Touriga Nacional showed the largest lesions while Aragonez recorded the smallest lesions amongst the whole lot of cultivars inoculated with Lasiodiplodia spp.. In general, Portuguese isolates were more aggressive than those from Peru, which demonstrated to be mildly aggressive. These results give a first insight on the response of selected Portuguese cultivars to Lasiodiplodia species, which are present in Portugal, but not commonly associated with Botryosphaeria dieback. This contributes to improve knowledge of the impact that Botryosphaeria dieback causal agents have on crucial national cultivars, which may help winegrowers not only to manage current cultural practices, but also to optimize decision making when planning the establishment of new vineyards.

Comparison of the infection biology of Teratosphaeria destructans and Teratosphaeria epicoccoides on Eucalyptus

Leaf blight caused by Teratosphaeria destructans is one of the most important diseases of Eucalyptus planted in the sub-tropics and tropics. In contrast, the better-known Teratosphaeria epicoccoides, while also a primary pathogen of Eucalyptus, causes less damage to trees in these areas. Although T. destructans is an aggressive pathogen, nothing is known about its infection biology. In this study, the conditions for infection and disease development caused by T. destructans and T. epicoccoides were evaluated and compared on a Eucalyptus grandis x Eucalyptus urophylla hybrid clone. The optimal temperature for the germination of T. destructans ranged from 25 to 30 oC and 15 to 20 oC for T. epicoccoides. The germination of these pathogens was favored under conditions of light and high levels of relative humidity. Penetration by T. destructans and T. epicoccoides occurred via stomata and the hyphae colonized the intercellular spaces of infected leaves. Symptoms were clearly visible three weeks after inoculation by both pathogens and reproductive structures started to develop in substomatal cavities at four weeks after inoculation. The results of this study will facilitate the establishment of rapid screening trials based on artificial inoculations aimed at reducing the impact of disease caused by T. destructans.

Cucumis melo L. Germplasm in Tunisia: Unexploited Sources of Resistance to Fusarium Wilt

Breeding for disease resistance has been one of the most important research objectives in melon for the last few decades. Fusarium wilt caused by Fusarium oxysporum f. sp. melonis (Fom) is among the most threatening melon fungal diseases along the Mediterranean coast, affecting yield and quality. Since genetic resistance is one of the best sustainable strategies that can be used to control this pathogen, 27 Tunisian melon accessions collected from local farmers have been tested using phenotypic and molecular approaches to identify new sources of resistance to be used directly as cultivars in affected areas or as resistance donors in breeding programs. The phenotypic evaluations, using artificial inoculations, showed several resistant accessions to the pathogenic races of Fom. Additionally, molecular analysis revealed that 13 out of 27 accessions carried the resistance Fom-1 gene (7 in homozygous state and 6 in heterozygous state), confirming their resistance to races 0 and 2. Two of them were also identified as heterozygous for the Fom-2 gene, being resistant to races 0 and 1. Furthermore, two accessions with a high level of resistance to the most virulent race 1.2 have been also reported. This melon germplasm should be explored as a potential source of resistance genes in breeding programs to develop new resistant melon cultivars.

First report of Phaeoacremonium oleae and P. viticola associated with olive trunk diseases in Italy

Over 300 trunk, branch and stem samples with vascular discolouration, necrotic wood and shoot death were collected from olive (Olea europaea) orchards in Lecce, Brindisi, Bari and Foggia provinces (Apulia region, Italy) from October to May from 2013 to 2019. Small chips of symptomatic wood samples were surface sterilised (5% NaOCl, 3 min; 70% ethanol, 30 s), rinsed (sterile distilled water, ×3), and placed onto potato dextrose agar (PDA) plates amended with 500 ppm streptomycin sulphate. After 14 days at 25 °C in the dark, hyphal tips of growing fungi, including different taxa, for instance Phaeoacremonium and Botryosphaeriaceae spp., were transferred to new PDA plates and incubated until sporulation. Monoclonal colonies resembling Phaeoacremonium-like genus (Mostert et al. 2006) were selected for further study, and genomic DNA of 59 representative isolates was extracted (Carlucci et al. 2013). Partial actin and β-tubulin genes were amplified with primers ACT-513F/ACT-783R (Carbone & Kohn 1999), and T1(O’Donnell & Cigelnik 1997) and Bt2b (Glass & Donaldson 1995), respectively. The sequenced amplicons were compared by BLAST algorithms with reference strains of Phaeoacremonium spp. retrieved from GenBank. Forty-four isolates showed 99% to 100% similarity with reference strains P. italicum, P. minimum, P. parasiticum, P. scolyti and P. sicilianum (Carlucci et al. 2015), nine with P. oleae, and six with P. viticola. Actin and β-tubulin sequences of P. oleae (Pm14) and P. viticola (Pm34) were submitted to GenBank (MW714561, MW714563; MZ318697, MZ318696). Microscopy of P. oleae isolates showed: conidiophores branched and unbranched, (18.7–)21.9–57.1(–67.8) × (2.9–)3.3–4.7(–5.2) (mean, 38.9×4.1) μm (n=30); conidia oblong-ellipsoidal to obovoid or subcylindrical 3.4 to 5.5 μm long, and 1.5 to 2.4 (mean, 4.6 × 2.2) μm wide (n=30). Microscopy of P. viticola isolates showed: conidiophores subcylindrical, branched at base (6.7–)8.9–27.2(–29.3) × (2.0–)2.6–3.3(–3.7) (mean, 21.4 × 3.2) μm (n=30); conidia oblong-ellipsoidal to obovoid or subcylindrical 3.3 to 6.8 μm long, and 1.1 to 2.2 (mean, 4.2 × 1.6) μm wide (n=30). In spring 2020, artificial inoculations were carried out with P. oleae (Pm14, Pm46) and P. viticola (Pm34, Pm43) strains on 10 healthy, 2-year-old olive seedlings cultivar ‘Coratina’. Agar plugs (diameter, 0.3–0.5 cm) from 10-day-old cultures grown on water agar at 23 (±2) °C were inserted under the bark of small wounds in the stems (length, 0.4–1.0 cm) made with a sterile scalpel. After inoculation, the wounds were wrapped with wet sterile cotton wool and sealed with Parafilm. Ten control olive seedlings were inoculated with sterile agar plugs. The experiment was replicated three times. All inoculated young olive plants were grown in pots in a greenhouse without temperature control. After 120 days, inoculated plants showed decline symptoms, and when cut longitudinally, brown streaks were observed in the wood. For P. oleae these streaks measured 3.0-5.5 cm long (standard deviation [SD], 0.9 cm, and for P. viticola they were 1.8-3.5 cm (SD, 0.62). Both fungal species were re-isolated from the symptomatic wood from 85% and 80%, respectively, of these inoculated olive seedlings, fulfilling Koch’s postulates. No symptoms were observed from olive seedlings used as control. P. oleae was first described as a fungal pathogen of wild olive (Olea europaea subsp. cuspidate) in South Africa by Spies et al. (2018), and P. viticola as a fungal pathogen of grapevine in France by Dupont et al. (2000). To the best of our knowledge, this is the first report of P. oleae associated with olive trunk disease in Italy, and the first report of P. viticola associated with olive trunk disease worldwide. References: Carbone I. & Kohn L.M. 1999. Mycologia 91:553. Carlucci A. et al. 2015. Eur. J. Plant Pathol. 141:717. Carlucci A. et al. 2013. Phytopathol. Mediterr. 52:517. Dupont et al. 2000. Mycologia 92:499-504. Glass N. L. & Donaldson G. C. 1995. J. Cl. Microbiol. 41: 1332. Mostert L. et al. 2006. Stud. Mycol. 54:1. O’Donnell K. & Cigelnik E. 1997. Mol. Phylogenetics Evol 7:103. Spies et al. 2018. Persoonia 40:26.

Bot Gummosis of Lemon (Citrus × limon) Caused by Neofusicoccum parvum

Neofusicoccum parvum, in the family Botryosphaeriaceae, was identified as the causal agent of bot gummosis of lemon (Citrus × limon) trees, in the two major lemon-producing regions in Italy. Gummy cankers on trunk and scaffold branches of mature trees were the most typical disease symptoms. Neofusicoccum parvum was the sole fungus constantly and consistently isolated from the canker bark of symptomatic lemon trees. It was identified on the basis of morphological characters and the phylogenetic analysis of three loci, i.e., the internal transcribed spacer of nuclear ribosomal DNA (ITS) as well as the translation elongation factor 1-alpha (TEF1) and β-tubulin (TUB2) genes. The pathogenicity of N. parvum was demonstrated by wound inoculating two lemon cultivars, ‘Femminello 2kr’ and ‘Monachello’, as well as citrange (C. sinensis × Poncirus trifoliata) ‘Carrizo’ rootstock. In artificial inoculations, the fungus was very aggressive on lemons and weakly virulent on citrange, consistently with symptoms observed in the field as a consequence of natural infections. This is the first report of N. parvum, both in a wide and in a strict taxonomic sense, as a pathogen of lemon in Italy.

Bio-chemical properties and susceptibility to fire blight (Erwinia amylovora Burrill) of scab-resistant apple cultivars (Malus domestica Borkh.)

The focus of our research was to evaluate different apples cultivars in terms of their biological properties and bioactive compounds content, and determine the levels of their resistance (or susceptibility) to fire blight. The properties of 10 scab-resistant apple cultivars were examined on the Žiča monastery estate (West Serbia) during the period from 2011 to 2015. The biological and chemical properties such as firmness, maturity stage, total soluble solids, total acids, total and reducing sugars, ascorbic acid content and surface blush of apple fruits were monitored. Various phenolic compounds in the tested samples were tentatively identified by LC-MS analyses. A study of generative properties included: number of flower buds, fruit mass and width, crop load, yield efficiency and yield. During the period of blooming and intensive shoot growth, artificial inoculations were carried out. For each cultivar, a fire blight score was determined by dividing the average length of necrotic tissue by the average total shoot length. In the study period, the cultivars ‘GoldRush’ (41.1 t ha−1) and ‘Florina’ (35.9 t ha−1) produced the highest yields, and the cultivars ‘Discovery’ (19.0 t ha−1) and Selection 25/63 (15.1 t ha−1) the lowest. The cultivar ‘William’s Pride’ produced the largest fruits, with an average fruit mass of 206.8 g. The earliest harvest period was recorded for the cultivar ‘Discovery’ (end of July), and the latest for the cultivar ‘GoldRush’ (beginning of October). The cultivar ‘Enterprise’ had the highest value of total phenols (432.2 mg 100 g −1 FW), while the cultivar ‘Topaz’ had the highest value of total flavanols (145.2 mg 100 g−1 FW). The highest degree of susceptibility to fire blight was found in Selection 25/63, and the greatest resistance was manifested by the cultivar ‘GoldRush’. In the five-year study period, the cultivars ‘GoldRush’, ‘Rewena’ and ‘Enterprise’ exhibited better bio-chemical properties and higher levels of resistance to fire blight than the remaining cultivars.

Coccinonectria pachysandricola, Causal Agent of a New Foliar Blight Disease of Sarcococca hookeriana

Woody plants of the Buxaceae, including species of Buxus, Pachysandra, and Sarcococca, are widely grown evergreen shrubs and groundcovers. Severe leaf spot symptoms were observed on S. hookeriana at the U.S. National Arboretum in Washington, DC, in 2016. Affected plants were growing adjacent to P. terminalis exhibiting Volutella blight symptoms. Fungi isolated from both hosts were identical based on morphology and multilocus phylogenetic analysis and were identified as Coccinonectria pachysandricola (Nectriaceae, Hypocreales), causal agent of Volutella blight of Pachysandra species. Pathogenicity tests established that Co. pachysandricola isolated from both hosts caused disease symptoms on P. terminalis and S. hookeriana, but not on B. sempervirens. Artificial inoculations with Pseudonectria foliicola, causal agent of Volutella blight of B. sempervirens, did not result in disease on P. terminalis or S. hookeriana. Wounding enhanced infection by Co. pachysandricola and Ps. foliicola on all hosts tested but was not required for disease development. Genome assemblies were generated for the Buxaceae pathogens that cause Volutella diseases: Co. pachysandricola, Ps. buxi, and Ps. foliicola; these ranged in size from 25.7 to 28.5 Mb. To our knowledge, this foliar blight of S. hookeriana represents a new disease for this host and is capable of causing considerable damage to infected plants.

Identification, pathogenicity and distribution of the causal agents of dieback in avocado orchards in Spain

An increased incidence of dieback from branches in several avocado orchards in southern Spain was observed in 2014. Surveys were conducted from May to October 2014, sampling the affected branches to isolate the causal agents. A total of 68 fungal isolates, recovered from ten avocado orchards, were identified, by morphological characterisation and DNA sequencing, as belonging to the genera: Neofusicoccum parvum (50%), Colletotrichum gloeosporioides (17.6%), Neofusicoccum luteum (16.2%), Neofusicoccum australe (13.2%), Neofusicoccum mediterraneum (1.5%) and Lasiodiplodia theobromae (1.5%). A decreasing level of virulence in artificial inoculations on avocado plants was observed in N. parvum, N. luteum, N. mediterraneum, N. australe, C. gloeosporioides and L. theobromae, there were significant differences among N. parvum and the rest of species of this genus, and significant differences were only observed between N. luteum and C. gloeosporioides. The geographical distribution of N. parvum and N. Luteum covers different areas, while C. gloeosporioides and N. australe are located only in the areas around Benamocarra and Vélez-Málaga (southern Spain), while N. mediterraneum and L. theobromae appear only occasionally. This is the first study of avocado branch cankers in Spain which identifies the causal agents and establishes their pathogenicity groups, with N. parvum as the most important causal agent of avocado dieback in this area.

First Extensive Microscopic Study of Butternut Defense Mechanisms Following Inoculation with the Canker PathogenOphiognomonia clavigignenti-juglandacearumReveals Compartmentalization of Tissue Damage

Ophiognomonia clavigignenti-juglandacearum endangers the survival of butternut (Juglans cinerea) throughout its native range. While screening for disease resistance, we found that artificial inoculations of 48 butternut seedlings with O. clavigignenti-juglandacearum induced the expression of external symptoms, but only after a period of dormancy. Before dormancy, compartmentalized tissues such as necrophylactic periderms (NPs) and xylem reaction zones (RZs) contributed to limiting pathogen invasion. Phenols were regularly detected in RZs, often in continuity with NPs during wound closure, and confocal microscopy revealed their presence in parenchyma cells, vessel plugs and cell walls. Vessels were blocked with tyloses and gels, particularly those present in RZs. Suberin was also detected in cells formed over the affected xylem by the callus at the inoculation point, in a few tylosis walls, and in longitudinal tubes that formed near NPs. Following dormancy, in all inoculated seedlings but one, defensive barriers were breached by O. clavigignenti-juglandacearum and then additional ones were produced in response to this new invasion. The results of this histopathological study indicate that trees inoculated in selection programs to test butternut canker resistance should go through at least one period of dormancy and that asymptomatic individuals should be dissected to better assess how they defend themselves against O. clavigignenti-juglandacearum.