Biological Control of Severe Fungal Phytopathogens by Streptomyces albidoflavus Strain CARA17 and Its Bioactive Crude Extracts on Lettuce Plants.

Lettuce crop is an important horticultural crop of several Mediterranean countries, including Italy. The Italian region which is a major producer of lettuce crops is Apulia, where this crop is cultivated in open fields an in greenhouses. Since several microbial pathogens are responsible for important diseases found on lettuce produced in greenhouses, in this study, the experimental activities focused on the most severe fungal soilborne pathogens, i.e., Sclerotinia sclerotiorum and Athelia rolfsii. Their control is often performed with fungicides which cause public concern over the environment and human health. The main aims of this study were to determine the biocontrol efficacy of a Streptomyces strain in vitro and in vivo conditions on lettuce seedlings against Athelia rolfsii and Sclerotinia sclerotiorum as severe fungal soilborne pathogens through the application of its vegetative propagules and putative bioactive crude extracts via filtrate culture. The results obtained confirm a significant effectiveness of CARA17 strain to control the severity of both fungal soilborne pathogens during two different experiments: when it is used as vegetative propagules and as a culture filtrate containing putative bioactive metabolites in vitro and in vivo conditions. These preliminary results demonstrated that the actinomycetes CARA17 strain is valid as a biocontrol agent (BCA) against both the severe phytopathogens used in this study. The biocontrol action performed from the CARA17 strain is clearly and mainly due to the putative bioactive crude extracts produced, but further studies are necessary to identify which metabolites (polyphenols, terpenes, fatty acids, etc.) are produced from this Streptomyces strain.

Correction: Mourou et al. Brassicaceae Fungi and Chromista Diseases: Molecular Detection and Host-Plant Interaction. Plants 2023, 12, 1033.

The authors wish to make the following corrections to this paper [...].

Brassicaceae Fungi and Chromista Diseases: Molecular Detection and Host­Plant Interaction

Brassicaceae plants cover a large number of species with great economic and nutritional importance around the world. The production of Brassica spp. is limited due to phytopathogenic fungal species causing enormous yield losses. In this scenario, precise and rapid detection and identification of plant-infecting fungi are essential to facilitate the effective management of diseases. DNA-based molecular methods have become popular methods for accurate plant disease diagnostics and have been used to detect Brassicaceae fungal pathogens. Polymerase chain reaction (PCR) assays including nested, multiplex, quantitative post, and isothermal amplification methods represent a powerful weapon for early detection of fungal pathogens and preventively counteract diseases on brassicas with the aim to drastically reduce the fungicides as inputs. It is noteworthy also that Brassicaceae plants can establish a wide variety of relationships with fungi, ranging from harmful interactions with pathogens to beneficial associations with endophytic fungi. Thus, understanding host and pathogen interaction in brassica crops prompts better disease management. The present review reports the main fungal diseases of Brassicaceae, molecular methods used for their detection, review studies on the interaction between fungi and brassicas plants, and the various mechanisms involved including the application of omics technologies.

Streptomyces albidoflavus Strain CARA17 as a Biocontrol Agent against Fungal Soil-Borne Pathogens of Fennel Plants.

Fennel crop is a horticultural plant susceptible to several soil-borne fungal pathogens responsible for yield losses. The control of fungal diseases occurring on fennel crops is very difficult with conventional and/or integrated means; although several chemical fungicides are able to contain specific fungal diseases, they are not registered for fennel crops. The intensive use of some fungicides causes public concern over the environment and human health. The main aims of this study were to assess the ability of a strain of Streptomyces albidoflavus CARA17 to inhibit the growth of fungal soil-borne pathogens, and to protect fennel plants against severe fungal soil-borne pathogens such as Athelia rolfsii, Fusarium oxysporum, Plectosphaerella ramiseptata, Sclerotinia sclerotiorum and Verticillium dahliae. This study confirmed that the CARA17 strain has been able to inhibit the mycelium growth of pathogens in vitro conditions with significant inhibition degrees, where S. sclerotiorum resulted in being the most controlled. The strain CARA17 was also able to significantly protect the fennel seedlings against fungal soil-borne pathogens used in vivo conditions, where the treatment with an antagonist strain by dipping resulted in being more effective at limiting the disease severity of each fungal soil-borne pathogen. Moreover, any treatment with the CARA17 strain, carried out by dipping or after transplanting, produced benefits for the biomass of fennel seedlings, showing significant effects as a promoter of plant growth. Finally, the results obtained showed that CARA17 is a valid strain as a biocontrol agent (BCA) against relevant fungal soil-borne pathogens, although further studies are recommended to confirm these preliminary results. Finally, this study allowed for first time worldwide the association of Plectosphaerella ramiseptata with fennel plants as a severe pathogen.

Untargeted and Targeted LC-MS/MS Based Metabolomics Study on In Vitro Culture of Phaeoacremonium Species.

Grapevine (Vitis vinifera L.) can be affected by many different biotic agents, including tracheomycotic fungi such as Phaeomoniella chlamydospora and Phaeoacremonium minimum, which are the main causal agent of Esca and Petri diseases. Both fungi produce phytotoxic naphthalenone polyketides, namely scytalone and isosclerone, that are related to symptom development. The main objective of this study was to investigate the secondary metabolites produced by three Phaeoacremonium species and to assess their phytotoxicity by in vitro bioassay. To this aim, untargeted and targeted LC-MS/MS-based metabolomics were performed. High resolution mass spectrometer UHPLC-Orbitrap was used for the untargeted profiling and dereplication of secondary metabolites. A sensitive multi reaction monitoring (MRM) method for the absolute quantification of scytalone and isosclerone was developed on a UPLC-QTrap. Different isolates of P. italicum, P. alvesii and P. rubrigenum were grown in vitro and the culture filtrates and organic extracts were assayed for phytotoxicity. The toxic effects varied within and among fungal isolates. Isosclerone and scytalone were dereplicated by matching retention times and HRMS and MS/MS data with pure standards. The amount of scytalone and isosclerone differed within and among fungal species. To our best knowledge, this is the first study that applies an approach of LC-MS/MS-based metabolomics to investigate differences in the metabolic composition of organic extracts of Phaeoacremonium species culture filtrates.

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 ß-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 ß-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.

First report of Phaeoacremonium amygdalinum associated with almond dieback and wood disease in Italy.

In the last 10 years, almond (Prunus dulcis (Mill.) Webb) cultivation areas in the region of Apulia (southern Italy) have increased. A recent survey in young almond orchards recorded characteristic disease symptoms including rapid collapse of branches, chlorosis of leaves (with sudden wilting and death) in June/July, and bud and shoot dieback in December-March. Internal wood symptoms on stems consisted of brown vascular streaking that led to death of young apical shoots within a few weeks. Wood samples were collected and taken to the laboratory, where they underwent disinfection according to Fisher et al. (2002). Fungal isolation techniques were applied, with small pieces of wood tissue (0.5-1.0 cm) placed on potato dextrose agar (PDA; Oxoid Ltd.) supplemented with 300 mg/L streptomycin sulphate, and incubated at 23 ±2 °C in the dark. After 10 days, based on morphological features carried out by microscopic studies, 85% (n = 55) of the resulting fungal colonies (N = 65) were preliminarily attributed to known fungal pathogens of woody tissues, of which 12.4% (n = 8) belonged to the Phaeoacremonium genus. These last were purified by transferring single germinating conidia onto fresh PDA plates. In particular, the conidiophores were mostly short, usually unbranched, erect to flexuous, up to 5-septate, brown to pale brown, verruculose on the lower part, (12-)15.7-41(-55) (av. -29) µm long and 1.6-3.2 (av. 2.1) µm wide (n = 30). Conidia hyaline, oblong ellipsoidal-obovoid or subcylindrical (3-)4-5 µm long and 1.5-3 (av. = 4.4 × 2.1) µm wide (n =30). All Phaeoacremonium isolates were successfully subjected to genomic DNA extraction, according to Carlucci et al. (2013). Partial actin and ß-tubulin genes were amplified with the ACT-513F/ACT-783R (Carbone and Kohn 1999) and T1 (O'Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) primers, respectively. Consensus sequences were compared with reference strains (extype) in the GenBank database, using the Basic Local Alignment Search Tool (BLAST). In particular, all isolates showed 99-100% similarity with reference strains of Phaeoacremonium amygdalinum Gramaje, Armengol & L. Mostert (JN191301; JN191305). Actin and ß-tubulin sequences of P. amygdalinum strain Pm10 were deposited in GenBank (MW716265; MW714562). In spring 2020, artificial inoculations with two isolates of P. amygdalinum (Pm10; Pm15) were carried out on 10 healthy, 2-year-old almond seedlings of cv. 'Filippo Cea'. Agar plugs (diameter, 0.3-0.5 cm) taken from 10-day-old cultures grown on PDA at 23 ±2 °C were inserted into small wounds under the bark of 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 almond seedling were used as controls, being inoculated with sterile agar plugs. The experiment was replicated three times. The inoculated young almond plants were grown in pots in a greenhouse without temperature control. After 150 days, the inoculated plants showed decline symptoms and internal longitudinal brown wood streaking (average, length, 2.7-6.4 cm). Phaeoacremonium amygdalinum was reisolated from symptomatic wood of 95% of the experimental almond seedlings, thus fulfilling Koch's postulates. No symptoms were observed on almond seedling used as controls. Phaeoacremonium amygdalinum was first described as a fungal pathogen of almond in Spain (Gramaje et al., 2012). To the best of our knowledge, this is the first report of P. amygdalinum associated with almond dieback disease in Italy. References Carbone I. and Kohn L.M. 1999. Mycologia 91:553. Carlucci A. et al. 2013. Phytopathol. Mediterr. 52:517. Fisher et al 2002. Mycol. Prog. 1:315 Glass N. L. and Donaldson G. C. 1995. J. Cl. Microbiol. 41:1332. Gramaje et al. 2012. Persoonia 28:1. O'Donnell K. and Cigelnik E. 1997. Mol. Phylogenetics Evol 7:103.

HPLC-HRMS Global Metabolomics Approach for the Diagnosis of "Olive Quick Decline Syndrome" Markers in Olive Trees Leaves.

Olive quick decline syndrome (OQDS) is a multifactorial disease affecting olive plants. The onset of this economically devastating disease has been associated with a Gram-negative plant pathogen called Xylella fastidiosa (Xf). Liquid chromatography separation coupled to high-resolution mass spectrometry detection is one the most widely applied technologies in metabolomics, as it provides a blend of rapid, sensitive, and selective qualitative and quantitative analyses with the ability to identify metabolites. The purpose of this work is the development of a global metabolomics mass spectrometry assay able to identify OQDS molecular markers that could discriminate between healthy (HP) and infected (OP) olive tree leaves. Results obtained via multivariate analysis through an HPLC-ESI HRMS platform (LTQ-Orbitrap from Thermo Scientific) show a clear separation between HP and OP samples. Among the differentially expressed metabolites, 18 different organic compounds highly expressed in the OP group were annotated; results obtained by this metabolomic approach could be used as a fast and reliable method for the biochemical characterization of OQDS and to develop targeted MS approaches for OQDS detection by foliage analysis.

Removal Ability and Resistance to Cinnamic and Vanillic Acids by Fungi.

Twelve fungal strains were assayed to investigate their resistance to cinnamic and vanillic acids and their ability to remove these compounds from a liquid medium. In a first step, the effect of the two aromatic acids (1 g/L) on the fungal growth kinetic was studied. The results were modelled through a logistic like function (Dantigny equation) to estimate τ, which is the time to the half-maximum colony diameter. The key findings of this part were as follows: (i) generally, cinnamic acid exerted a stronger effect than vanillic acid; (ii) aromatic acids exerted a delay on the growth of some fungi and only one strain (Athelia rolfsii) was completely inhibited. In the second part, fungi were assayed to investigate their ability to remove cinnamic and vanillic acids (ca. 350 mg/kg) from liquid media at pH 3.5. The results indicated that the most efficient fungi were Aspergillus niger and Lasiodiplodia theobromae.

A Comparative Study on Trichoderma harzianum and a Combination of Candida/Bacillus as Tools for the Bioremediation of Table Olive Processing Water.

A comparative study was performed on Trichoderma harzianum and a combination of Candida boidinii/Bacillus pumilus to reduce the polluting effect of TOPW (Table Olive Processing Water) from the Spanish style. A 2k fractional design was used to study the effect of pH (6-11 for the fungus and 6-9 for Candida/Bacillus), temperature (10-35 °C) and duration (7-14 days for Candida/Bacillus and 14-21 days for T. harzianum), and the effect on phenol reduction, COD and color was evaluated. The experiments were also performed on diluted TOPW (dilution ratio 1:1). Generally, Trichoderma removed higher amounts of phenols and reduced COD more than the combination Candida/Bacillus, thus confirming the higher efficiency of filamentous fungi reported in the literature. The dilution of TOPW had an effect only on COD reduction; however, the effect was mild, at least for T. harzianum (4%), while yield increase was 9% for Bacillus/Candida. pH acted in a different way on phenol removal and COD reduction; an increase of pH caused a reduction of efficiency for COD, while the effect was positive for phenols.

Evaluation of Fungal Growth on Olive-Mill Wastewaters Treated at High Temperature and by High-Pressure Homogenization.

Reuse of olive mill wastewaters (OMWWs) in agriculture represents a significant challenge for health and safety of our planet. Phytotoxic compounds in OMWW generally prohibit use of untreated OMWWs for agricultural irrigation or direct discharge into surface waters. However, pretreated OMWW can have positive effects on chemical and microbiological soil characteristics, to fight against fungal soil-borne pathogens. Low amounts of OMWW following thermal (TT-OMWW) and high-pressure homogenization (HPH-OMWW) pretreatments counteracted growth of some of 12 soil-borne and/or pathogenic fungi examined. With fungal growth measured as standardized change in time to half maximum colony diameter, Δτ, overall, HPH-OMWW showed increased bioactivity, as increased mean Δτ from 3.0 to 4.8 days. Principal component analysis highlighted two fungal groups: Colletotrichum gloeosporioides, Alternaria alternata, Sclerotium rolfsii, and Rosellinia necatrix, with growth strongly inhibited by the treated OMWWs; and Aspergillus ochraceus and Phaeoacremonium parasiticum, with stimulated growth by the treated OMWWs. As a non-thermal treatment, HPH-OMWW generally shows improved positive effects, which potentially arise from preservation of the phenols.

Fungal bioremediation of olive mill wastewater: using a multi-step approach to model inhibition or stimulation.

BACKGROUND: Olive mill wastewaters (OMWWs) possess a strong environmental impact; the use of fungi as tools for bioremediation could be a promising method. RESULTS: Twenty-nine fungi were grown on minimal media supplemented with five different kinds of OMWWs (5-15%). Radial growth was assessed for 21 days and the data were modelled through the Dantigny-logistic like function to estimate τ, i.e. the time to attain half of the maximum diameter. Growth on potato dextrose agar and water agar (WA, minimal medium without supplementation) was used as reference. The differences in τ between PDA/WA and minimal media with OMWWs were modelled through a multi-factorial ANOVA, using the concentration of OMWW, the kind of wastes and fungi as categorical predictors. Finally, a principal component analysis was run to group and divide resistant and sensitive fungi. Some fungi experienced a positive Δτ, thus suggesting an inhibition by OMWW, whereas other isolates were enhanced. CONCLUSIONS: Some isolates (for example Aspergillus ochraceus) showed a promising trend and could be possible candidates for a validation on a real scale. © 2016 Society of Chemical Industry.

Charcoal Canker of Pear, Plum, and Quince Trees Caused by Biscogniauxia rosacearum sp. nov. in Southern Italy.

The genus Biscogniauxia is paraphyletic to members of the family Xylariaceae and includes at least 52 species to date that are mainly pathogens of dicotyledonous angiosperm trees. Most of these are forest trees, such as those in the genera Acacia, Acer, Alnus, Eucalyptus, Fraxinus, Populus, and Quercus, and other species of minor importance. Biscogniauxia spp. have been reported as endophytes or secondary invaders that attack only stressed plants. During a survey in rosaceous orchards in southern Italy, several charcoal cankers were observed and stroma samples were collected. A collection of 31 Biscogniauxia isolates was analyzed. Their phylogenetic relationships were determined through study of the internal transcribed spacer, ß-tubulin, and actin gene sequences. Combining morphological, cultural, and molecular data, a new species of Biscogniauxia is described here as Biscogniauxia rosacearum. This new species was isolated for the first time from rosaceous hosts in Apulia. Pathogenicity tests showed that it causes symptoms on stems when artificially inoculated and produces stromata on the bark surface.

Characterization of Botryosphaeriaceae Species as Causal Agents of Trunk Diseases on Grapevines.

Botryosphaeriaceae spp. have a cosmopolitan distribution and a wide range of plant hosts. Over the last 15 years, worldwide, 21 species of this family have been associated with grapevine trunk diseases that cause cankers and dieback on grapevines. Here, we surveyed vineyards of Vitis vinifera 'Lambrusco', 'Sangiovese', and 'Montepulciano' in three areas of the Foggia province (Cerignola, Foggia, and San Severo) in southern Italy. Wood samples from grapevines showing general decline, dieback, cankers, and wood and foliar discoloration yielded 344 fungal isolates identified as Botryosphaeriaceae spp. A phylogenetic study combining internal transcribed spacer and translation elongation factor 1-α sequences of 60 representative isolates identified nine botryosphaeriaceous species: Botryosphaeria dothidea, Diplodia corticola, D. mutila, D. seriata, Dothiorella iberica, Do. sarmentorum, Lasiodiplodia citricola, L. theobromae, and Neofusicoccum parvum. Pathogenicity tests confirmed that all nine species cause canker and dieback of grapevines. However, this is the first report of L. citricola as causal agent of wood cankers and dieback of grapevine. To date, including L. citricola, there are 25 botryosphaeriaceous species associated with V. vinifera worldwide, of which 12 have been reported for grapevines in Italy.

Phaeoacremonium italicum sp. nov., associated with esca of grapevine in southern Italy.

To date at least 42 Phaeoacremonium species are known throughout the world. These fungal pathogens are responsible for several syndromes that occur in wood of different hosts, 27 of which have been associated with decline and dieback diseases or esca of grapevine and have been abundantly isolated from necrotic wood of grapevines with Petri and esca disease in vineyards worldwide. During a survey carried out in five vineyards of the grapevine cultivar Italia, several symptomatic samples were collected. A collection of 28 Phaeoacremonium isolates was analyzed. The phylogenetic relationships of the isolates were determined through the study of the ß-tubulin and actin gene sequences. Combining morphological, culture and molecular data, three known Phaeoacremonium spp. were found, namely Pm. aleophilum, Pm. parasiticum and Pm. scolyti. One new species is described. Phaeoacremonium italicum can be identified by the common occurrence of bundles of up to 13, conidiophores with up to seven septa, occasionally branched, percurrent rejuvenation and predominantly phialides of type II. This novel species thus is isolated for the first time from grapevine in Apulia (southern Italy).