Amplicon metagenomics to identify and detect fungal The Fruit and oomycete plant Microbiome: A pathogens: potentialities New Frontier and shortcomings

Leonardo Schena, Ahmed Abdelfattah

Dipartimento di Agraria, Università Mediterranea di Reggio Calabria, Località Feo di Vito, 89122 Reggio Calabria, Italy Department of Ecology, Environment and Plant Sciences, Stockholm University, 106 91 Stockholm, Sweden Amplicon metagenomics Nilsson et al., 2018: Nature Reviews in Microbiology. or metabarcodings https://doi.org/10.1038/s4 1579-018-0116-y 454 pyrosequencing Ion Torrent PacBio Illumina MiSeq, HiSeq, NovaSeq MinION A huge potentiality: the only available technique that has the potential to detect the whole fungal diversity

doi:10.1128/microbiolspec.FUNK-0052-2016

Oomycota

…. the fungal species on the earth were estimated to be 12 (11.7–13.2) million State of the World’s Fungi 2018 https://stateoftheworldsfungi.org/2018/reports/SOTWFungi_2018_Full_Report.pdf According to the report of the Royal Botanical Garden (KEW) on the State of the Worl Fungi (2018) there are AT LEAST 2 MILLION SPECIES OF FUNGI YET TO BE DESCRIBED an immense ….. RESOURCE …. but also a THREAT The Kingdoms fungi and chromista contains the largest number of phytopatogenic microrganisms

State of the World’s Fungi 2018 https://stateoftheworldsfungi.org/2018/reports/SOTWFungi_2018_Full_Report.pdf Biosecurity issues Many species are harmless in their native environment and causes great losses only when introduced in a new environment

Harvested fruit and vegetables represent an important vector of plant pathogens

… Plant Protection Organizations focus their attention on already known potential threats ….. but future disease outbreaks are likely to be cause by still unknown plant pathogens! Guignardia citricarpa = Phyllosticta citricarpa How can we protect our fruit from hidden enemies?! Despite the huge potentialy of metabarcoding approches, their use to study fungal plant pathogens is still limited

High‐throughput identification technologies ……. have been only rarely used for monitoring and testing ecological hypotheses in plant pathogens and pests in spite of their immense importance in agriculture, forestry and plant community dynamics.

The use of HTS methods in mycopathology has somewhat lagged behind other fields of mycology despite the potential usefulness of HTS for surveillance. Much of the available information comes from studies that did not specifically address pathogens.

Why? Current methods do not enable a reliable identification of taxa at the species and/or sub- species level …… related fungal and oomycete species, with very similar or identical ITS regions, can have a completely different behaviour. Coffee Berry Disease

Weir et al., 2012 Studies in Mycology 73: 115–180.

Colletotrichum gloeosporioides species complex. Concatenated sequences: ACT, TUB2, CAL, CHS-1, GAPDH, GS, ITS, and SOD2 Our experience with metabarcoding or amplicon metagenomics to study fungal and oomycete plant pathogens - Analysis of fungal taxa (pathogens and non- pathogens) associate to different plant species Phylogenetic analyses to and organs increase the level of - Investigation of disease aetiology descrimination among species - Evaluation of the role of key insects as local and international vectors of plant pathogens - Investigate diffusion and diversity of Use of different of primers Phytophthora species - Broad range primers - Development and validation of qPCR detection - Genus specific primers methods

Future perspecitives PLoS ONE 10(7): e0131069. Sampling: May, June, October, December

Primers ITS3-ITS4 Aureobasidium Devriesia Pseudocercospora Cladosporium Colletotrichum Fusarium Aureobasidium Devriesia Pseudocercospora Cladosporium Colletotrichum Fusarium PSEUD 1 (5893) CLA 1 (2200) 61 Sequences64 of the most “important” (abundant) genera Other C. acutatum s.l. species A AY438249 P. cladosporioides CLA 2 (2447) were extracted AY438250 P. cladosporioides and “manually” analyzedC HM148003 along C. cladosporioideswith COLL 1 (141) 63 PSEUD 2 (33) HM147994 C. acalyphae 50 JQ948354 C. acutatum E validated PSEUD reference 3 (4) sequences HM147995 C. angustisporum P. cladosporioides JQ948373 C. acutatum C. acutatum s.s. PSEUD 4 (8) HM147998 C. asperulatum JQ948399 C. acutatum GU269678 P. cladosporioides HM147999 C. australiense PSEUD 1 (5893) CLA 1 (2200) 61 64 56 JQ948297 C. cuscutae Other C. acutatum s.l. species DQ303090 P. crousii A HM148000 C. AY438249 basiinflatum P. cladosporioides CLA 2 (2447) AY438250 P. cladosporioides C HM148003 C. cladosporioides JQ948307 C. fioriniae GU269653 P. araliae HM148001 C. chalastosporoides COLL 1 (141) 63 PSEUD 2 (33) HM147994 C. acalyphae JQ948470 C.salicis Source GU269654 of validated P. arecacearum reference FJ936159 C. colombiae 50 JQ948354 C. acutatum E PSEUD 3 (4) HM147995 C. angustisporum 72 COLL 2 (7729) JQ948373 C. acutatum 51 GU269696 P. dendrobii HM148081 C. PSEUD delicatulum 4 (8) HM147998 C. asperulatum sequences: COLL 3 (9393) JQ948399 C. acutatum GU269678 P. cladosporioides HM147999 C. australiense GU269774 P. rumohrae HM148090 C. exasperatum 56 JQ948297 C. cuscutae DQ303090 P. crousii HM148000 C. basiinflatum JQ948455 C. australe - GU269791Specific P. taxonomic plectranthi studies HM148091 C. exile JQ948307 C. fioriniae GU269653 P. araliae HM148001 C. chalastosporoides JQ948416 C. godetiae GU269652 P. araliae HM148092 C. flabelliforme JQ948470 C.salicis GU269654 P. arecacearum FJ936159 C. colombiae JQ948444 C. johnstonii - Reference Sequence (RefSeq) 72 COLL 2 (7729) AY725526 P. pseudoeucalyptorum HM14809451 GU269696 C. funiculosum P. dendrobii HM148081 C. delicatulum57 JQ948454 COLL 3 (9393) GU269774 P. rumohrae HM148090 C. exasperatum - UNITE GU269660 database P. balsaminae HM148095 C. gamsianum JQ948455 C. australe GU269791 P. plectranthi HM148091 C. exile C. phormii JQ948448 73 GU269695 P. dianellae HM148096 C. globisporum C. godetiae? JQ948416 C. godetiae GU269652 P. araliae HM148092 C. flabelliforme C. pyricola JQ948445 - Other specific fungal barcoding JQ948444 C. johnstonii GU269723 P. humuli HM148097 AY725526 C. hillianum P. pseudoeucalyptorum HM148094 C. funiculosum C. salicis JQ948478 57 JQ948454 GU269751 P. nephrolepidis HM148101 C. GU269660 inversicolor P. balsaminae HM148095 C. gamsianum 63databases C. acerbum JQ948459 C. phormii JQ948448 73 GU269695 P. dianellae HM148096 C. globisporum GU269787 P. profusa HM148110 C. iranicum C. pyricola JQ948445 GU269723 P. humuli HM148097 C. hillianum 63 C. rhombiforme JQ948457 21 GU269792 P. crocea HM148111 C. licheniphilum C. salicis JQ948478 - GenBank: reliability was GU269751 P. nephrolepidis HM148101 C. inversicolor 63 C. acerbum JQ948459 DQ303077 P. natalensis HM148115 GU269787 C. lycoperdinum P. profusa HM148110 C. iranicum evaluated according to 65 63 C. rhombiforme JQ948457 GU269780 P. natalensis 21 HM148117 GU269792 C. myrtacearum P. crocea HM1481110.005 C. licheniphilum GU269730 P. kiggelariae HM148120 DQ303077 C. paracladosporioidesP. natalensis HM148115 C. lycoperdinum different parameters, including 65 GU269734 P. longispora HM148121 GU269780 C. perangustumP. natalensis HM148117 C. myrtacearum 0.005 GU269730 P. kiggelariae HM148120 COLL C. paracladosporioides 4 (37) 71 the consistence GU269747 P. musaeof sequences JF499837 GU269734 C. phaenocomae P. longispora HM148121 C. perangustum COLL 5 (16) COLL 4 (37) AF362054 P. cordiana HM14815371 C. phyllactiniicola GU269747 P. musae JF499837 C. phaenocomae F 28 from different sources, AF362054 P. cordiana HM148153 JQ005175 C. phyllactiniicola C. karstii COLL 5 (16) F GQ852770 P. tereticornis 28 HM148154 C. phyllophilum 68 GQ852770 P. tereticornis HM148154 JQ005221 C. phyllophilum C. phyllanthi C. karstii68 JQ005175? C. karstii GU269650 P. acericolaavailable details on sequenced FJ936160 C. piniponderosae JQ005221 C. phyllanthi GU269650 P. acericola FJ936160 C. JQ005222piniponderosae C. annellatum GU269656 P. assamensis GU269656 HM148158 P. assamensis C. pseudocladosporioides HM148158 C. pseudocladosporioides JQ005222 C. annellatum GU269663 P. callicarpaeisolates, year of publication, GU269663 HM148193 P. callicarpae C. rectoides HM148193 C. rectoides Other C. boninense s.l. species Other C. boninense s.l. species GU269770 P. rhapisicola HM148195 C. scabrellum 66 GU269770 P. rhapisicola HM148195 C. scabrellum 66 JQ005236 C. dacrycarpi etc. etc. JQ324983 P. xanthocercidis EF679354 C. silenes JQ005236 C. dacrycarpi JQ005237 C. constrictum JQ324983 P. xanthocercidis EF679354 C. silenes HM148196 C. subuliforme Other Pseudocercospora species JQ005237 C. constrictum 86 JQ005238 C. constrictum HM148196 C. subuliforme HM14822486 C. varians Other Pseudocercospora species 5 JQ005238 C. constrictum JQ005152 C. gloeosporioides HM148224 C. varians HM148226 C. verrucocladosporioides 5 Other Pseudocercospora species JQ005152 C. gloeosporioides HM148226 C. verrucocladosporioides HM148230 C. xylophilum Other Pseudocercospora species 88 CLA 3 (7) 0.005 HM148230 C. xylophilum JF770450 C. grevilleae 0.005 0.01 88 CLA 3 (7) DQ780379 C. langeronii 63 DEV 1 (5976) JF770450 C. grevilleae 87 DQ780386 C. psychrotolerans G DEV 2 (18) 98 DEV 3 (11) 0.01 SPIL DQ780379 (75) C. langeronii 63 DEV 1 (5976) C. herbarum complex 51 DEV 4 (4) KF766166 Spilocaea oleagina 57 87 DQ780386 C. psychrotolerans B G DEV 2 (18) HQ599602 D. fraseriae EU035435 Fusicladium phillyreae HM14806798 C. colocasiae DEV 3 (11) JX294932 D. ficus 65 EU035434 Spilocaea oleagina SPIL (75) C. herbarum complex 83 HM148197 C. tenuissimum GU214634 D. lagerstroemiae AF338401 Spilocaea oleagina 51 DEV 4 (4) KF766166 Spilocaea oleagina 57 JN906978 C. aphidis 51 GU214633 D. hilliana B AF338400 Spilocaea oleagina HQ599602 D. fraseriae 93 C. sphaerospermum complex JF951148 D. queenslandica EU035435 Fusicladium phillyreae AF338403 HM148067 Spilocaea C. colocasiae oleagina DQ780374 C. salinae JX294932 D. ficus82 HQ599605 D. xanthorrhoeae 65 EU035434 Spilocaea oleagina 86 AF338402 Spilocaea oleagina 83 HM148197 C. tenuissimum GU214634 D. lagerstroemiae 38 JX069875 D. agapanthi AF338401 Spilocaea oleagina JN906978 C. aphidis AF338399 Spilocaea oleagina EU035438 Venturia tremulae 0.005 51 GU214633 D. hilliana 93 AF338400 Spilocaea oleagina 98 C. sphaerospermum KF793785 Venturia complex saliciperda JF951148 D. queenslandica0.02 AF338403 Spilocaea oleagina EU03546 Venturia pyrina DQ780374 C. salinae 82 HQ599605 D. xanthorrhoeae 86 AF338402 Spilocaea oleagina KF156040 Venturia inaequalis AUR 381 (125632) JX069875 D. agapanthi AF338399 Spilocaea oleagina AUR 2 (8) D 0.005 EU035438 Venturia tremulae 0.005 FJ150902 A. pullulans var. pullulans 98 KF793785 Venturia saliciperda FJ1509080.02 A. pullulans var. pullulans EU03546 Venturia pyrina FJ150900 A.pullulans var. pullulans AUR 3 (49) KF156040 Venturia inaequalis AUR 1 (125632) 95 FJ150875 A. pullulans var. namibiae FJ150886 A. pullulans var. melanogenum AUR 2 (8) D 80 FJ150893 A. pullulans var. subglaciale 0.005 FJ150902 A. pullulans var. pullulans

FJ150908 A. pullulans var. pullulans 0.005 FJ150900 A.pullulans var. pullulans AUR 3 (49) 95 FJ150875 A. pullulans var. namibiae FJ150886 A. pullulans var. melanogenum 80 FJ150893 A. pullulans var. subglaciale

0.005 PSEUD 1 (5893) CLA 1 (2200) 61 64 Other C. acutatum s.l. species A AY438249 P. cladosporioides CLA 2 (2447) AY438250 P. cladosporioides C HM148003 C. cladosporioides COLL 1 (141) 63 PSEUD 2 (33) HM147994 C. acalyphae 50 JQ948354 C. acutatum E PSEUD 3 (4) HM147995 C. angustisporum JQ948373 C. acutatum PSEUD 4 (8) HM147998 C. asperulatum JQ948399 C. acutatum GU269678 P. cladosporioides HM147999 C. australiense 56 JQ948297 C. cuscutae DQ303090 P. crousii HM148000 C. basiinflatum JQ948307 C. fioriniae GU269653 P. araliae HM148001 C. chalastosporoides JQ948470 C.salicis GU269654 P. arecacearum FJ936159 C. colombiae 72 COLL 2 (7729) 51 GU269696 P. dendrobii HM148081 C. delicatulum COLL 3 (9393) GU269774 P. rumohrae HM148090 C. exasperatum JQ948455 C. australe GU269791 P. plectranthi HM148091 C. exile JQ948416 C. godetiae PSEUD 1 (5893) CLA 1 (2200) 61 GU269652 P. araliae HM14809264 C. flabelliforme Other C. acutatum s.l. species A AY438249 P. cladosporioides CLA 2 (2447) JQ948444 C. johnstonii AY725526 P. pseudoeucalyptorum HM148094 AY438250 C. funiculosum P. cladosporioides C HM148003 C. cladosporioides 57 JQ948454 COLL 1 (141) 63 PSEUD 2 (33) 61 HM147994 C. acalyphae GU269660 P. balsaminae PSEUD 1 (5893) HM148095 CLA 1 C. (2200) gamsianum Other C. acutatum s.l. species 50 JQ948354 C. acutatum E 64 PSEUD 3 (4) HM147995 C. angustisporum C. phormii JQ948448 73 A AY438249 P. cladosporioides CLA 2 (2447) JQ948373 C. acutatum GU269695 P. dianellae C HM148096 PSEUD C. 4globisporum (8) HM147998 C. asperulatum AY438250 P. cladosporioides HM148003 C. cladosporioides C. pyricola JQ948445 JQ948399 C. acutatum GU269678 P. cladosporioides HM147999 COLL 1 C.(141) australiense GU269723 P. humuli 63 PSEUD 2 (33) HM148097 HM147994 C. C. hillianum acalyphae 50 E 56 JQ948297 C. cuscutae DQ303090 P. crousii JQ948354HM148000 C.C. acutatumbasiinflatum C. salicis JQ948478 PSEUD 3 (4) HM147995 C. angustisporum JQ948307 C. fioriniae 63 GU269751 P. nephrolepidis HM148101 GU269653 C. P.inversicolor araliae HM148001 JQ948373 C. chalastosporoides C. acutatum PSEUD 4 (8) HM147998 C. asperulatum C. acerbum JQ948459 JQ948470 C.salicis GU269787 P. profusa HM148110 GU269654 C. P.iranicum arecacearum FJ936159 JQ948399 C. colombiae C. acutatum GU269678 P. cladosporioides HM147999 C. australiense 72 COLL 2 (7729) 51 GU269696 P. dendrobii HM14808156 C.JQ948297 delicatulum C. cuscutae C. rhombiforme JQ948457 21 DQ303090 P. crousii HM148000 C. basiinflatum 63 COLL 3 (9393) GU269792 P. crocea HM148111 GU269774 C. P.licheniphilum rumohrae HM148090 C.JQ948307 exasperatum C. fioriniae GU269653 P. araliae HM148001 C. chalastosporoides JQ948455 C. australe GU269791 P. plectranthi HM148091 C. exile JQ948470 C.salicis DQ303077 P. natalensis GU269654 P. arecacearum HM148115 FJ936159 C. C. lycoperdinumcolombiae JQ948416 C. godetiae GU269652 P. araliae HM14809272 C. flabelliforme COLL 2 (7729) 51 GU269696 P. dendrobii 65 HM148081 C. delicatulum JQ948444 C. johnstonii GU269780 P. natalensis HM148117 AY725526 C. P.myrtacearum pseudoeucalyptorum HM148094 C. funiculosum COLL0.005 3 (9393) GU269774 P. rumohrae HM148090 C. exasperatum 57 JQ948454 GU269730 P. kiggelariae HM148120 GU269660 C. paracladosporioides P. balsaminae HM148095 C. gamsianum JQ948455 C. australe GU269791 P. plectranthi HM148091 C. exile C. phormii JQ948448 73 GU269695 P. dianellae HM148096 C. globisporum JQ948416 C. godetiae GU269734 P. longispora GU269652 P. araliae HM148121 HM148092 C. C. perangustum flabelliforme C. pyricola JQ948445 GU269723 P. humuli HM148097 C. hillianum JQ948444 C. johnstonii AY725526 P. pseudoeucalyptorum HM148094 C. funiculosum COLL 4 (37) C. salicis JQ948478 GU269747 P. musae JF499837 GU269751 C. phaenocomae P. nephrolepidis HM148101 C.57 inversicolor JQ948454 71 GU269660 P. balsaminae 63 HM148095 C. gamsianum C. acerbum JQ948459 GU269787 P. profusa HM148110 C. iranicum C. phormii COLL JQ948448 5 (16) 73 F C. rhombiforme JQ948457 AF362054 P. cordiana GU269695 P. dianellae HM148153 HM148096 C. C. phyllactiniicola globisporum 63 28 21 GU269792 P. crocea HM148111 C. licheniphilum C. pyricola JQ948445 GU269723 P. humuli HM148097 C. hillianum JQ005175 C. karstii GQ852770 P. tereticornis HM148154 DQ303077 P. C.natalensis phyllophilum HM148115 C. lycoperdinum C.68 salicis JQ948478 GU269751 P. nephrolepidis HM148101 C. inversicolor 65 GU269650 P. acericola 63 FJ936160 GU269780 P. C. natalensis piniponderosae HM148117 C. myrtacearum JQ005221 C. acerbum C. JQ948459phyllanthi 0.005 GU269787 P. profusa GU269730 P. kiggelariae HM148110 C. iranicum HM148120 C. paracladosporioides 63 C. rhombiforme JQ005222 JQ948457 C. annellatum GU269656 P. assamensis 21 GU269792 P. crocea HM148158 GU269734 HM148111 P. C.longispora C. pseudocladosporioides licheniphilum HM148121 C. perangustum DQ303077 P. natalensis HM148115 GU269747 C. lycoperdinum P. musae JF499837 C. phaenocomae COLL 4 (37) GU269663 P. callicarpae 71 HM14819365 C. rectoides Other C. boninense s.l. species GU269780 P. natalensis AF362054 P. cordiana HM148117 C. myrtacearum HM1481530.005 C. phyllactiniicola COLL 5 (16) F 28 66 GU269770 P. rhapisicola GU269730 P. kiggelariae HM148195 HM148120 C. C. scabrellum paracladosporioides 68 JQ005175 C. karstii GQ852770 P. tereticornis HM148154 C. phyllophilum JQ005236 C. dacrycarpi GU269734 P. longispora GU269650 P. acericola HM148121 C. perangustum FJ936160 C. piniponderosae JQ005221 C. phyllanthi JQ324983 P. xanthocercidis EF679354 C. silenes COLL 4 (37) JQ005222 C. annellatum 71 GU269747 P. musae GU269656 P. assamensis JF499837 C. phaenocomae HM148158 C. pseudocladosporioides JQ005237 C. constrictum AF362054 P. cordiana GU269663 P. HM148196 callicarpae HM148153 C. C. subuliforme phyllactiniicola HM148193 COLL C.5 (16)rectoides 86 F Other C. boninense s.l. species Other Pseudocercospora28 species JQ005238 C. constrictum GQ852770 P. tereticornis GU269770 P. rhapisicola HM148154 C. phyllophilum 68 HM148195 JQ005175 C. C. scabrellum karstii 66 5 HM148224 C. varians JQ005236 C. dacrycarpi JQ324983 P. xanthocercidis EF679354 JQ005221 C. C.silenes phyllanthi JQ005152 C. gloeosporioides GU269650 P. acericola FJ936160 C. piniponderosae JQ005237 C. constrictum HM148226 C. verrucocladosporioides HM148196 JQ005222 C. subuliforme C. annellatum GU269656 P. assamensis Other Pseudocercospora HM148158 species C. pseudocladosporioides 86 JQ005238 C. constrictum Other Pseudocercospora species HM148224 C. varians GU269663 P. callicarpae 5 HM148230 HM148193 C. C. xylophilum rectoides Other C. boninense s.l. species JQ005152 C. gloeosporioides GU269770 P. rhapisicola HM148195 C. scabrellum HM14822666 C. verrucocladosporioides Other Pseudocercospora88 CLA species 3 (7) 0.005 JQ005236 C. dacrycarpi JQ324983 P. xanthocercidis EF679354 C. silenes HM148230 C. xylophilum JQ005237 C. constrictum 0.005 HM148196 JF770450 C. subuliforme C. grevilleae 8688 CLA 3 (7) Other Pseudocercospora species JQ005238 JF770450 C. C. constrictum grevilleae 5 HM148224 C. varians 0.01 0.01 DQ780379 C. langeronii JQ005152 C. gloeosporioides63 DEV 1 (5976) Spilocaea HM148226oleagina C. verrucocladosporioides DQ780379 C. langeronii PSEUD 1 (5893) CLA63 1 (2200) DEV 1 (5976) 61 Other Pseudocercospora species 64 Other C. acutatum s.l. species HM148230 DQ780386 C. xylophilum C. psychrotolerans 87A DQ780386G C. psychrotolerans AY438249 P. cladosporioides GDEV 2 (18) CLA 2 (2447) DEV 2 (18) 87 AY438250 P. cladosporioides C HM148003 C. cladosporioides 0.005 98 DEV 3 (11) COLL 1 (141) 88 CLA 3 (7) 63 PSEUD 2 (33) 98 HM147994 C. acalyphae SPIL (75) Devriesia fraseriae DEV 3 (11) 50 JQ948354 C. acutatum E C. herbarum complexPSEUD 3 (4) 51 HM147995 DEVC. angustisporum 4 (4) SPIL (75) KF766166 Spilocaea JF770450 oleagina C. grevilleae 57 JQ948373 C. acutatum C. herbarum complexB PSEUD 4 (8) HM147998 C. asperulatum 0.01 51 DEV 4 (4) HQ599602 D. fraseriae JQ948399 C. acutatum EU035435 Fusicladium DQ780379 phillyreae C. langeronii 63 DEV 1 GU269678 (5976) P. cladosporioides HM147999 C. australiense KF766166 Spilocaea oleagina 57 HM148067 C. colocasiae JX294932 D. ficus 56 JQ948297 C. cuscutae B DQ780386 C. psychrotolerans DQ303090 DEV 2 P. (18) crousii HM148000 C. basiinflatum 65 EU035434 Spilocaea87 oleagina G HM148197 C. tenuissimum HQ599602 D. fraseriae JQ948307 C. fioriniae 83 GU269653 P. araliae GU214634 HM148001 D. lagerstroemiae C. chalastosporoides EU035435 Fusicladium phillyreae AF338401 Spilocaea oleagina 98 DEV 3 (11) JQ948470 C.salicis SPIL (75) HM148067 C. colocasiae JN906978 C. aphidis GU269654 P. arecacearum 51 FJ936159 GU214633 C. colombiae D. hilliana C. herbarum complex JX294932 D. ficus 72 COLL 2 (7729) AF338400 Spilocaea oleagina 51 DEV51 GU269696 4 (4) P. dendrobii HM148081 C. delicatulum 65 EU035434 Spilocaea oleagina 93 57 C. sphaerospermum complex JF951148 D. queenslandica COLL 3 (9393) KF766166 Spilocaea oleagina HM148197 C. tenuissimum GU269774 P. rumohrae HM148090 C. exasperatum B AF33840383 Spilocaea oleagina HQ599602 GU214634 D. fraseriae D. lagerstroemiae JQ948455 C. australe EU035435 Fusicladium phillyreae DQ780374 GU269791 C. salinae P. plectranthi 82 HQ599605 HM148091 C.D. exile xanthorrhoeae AF338401 Spilocaea oleagina AF338402 HM148067Spilocaea oleagina C. colocasiae JX294932 D. ficus JQ948416 C. godetiae 86 JN906978 C. aphidis GU269652 P. araliae HM148092 C. flabelliforme 65 EU035434 Spilocaea oleagina 51 GU214633 D. hilliana38 JX069875 D. agapanthi JQ948444 C. johnstonii AF33839983 SpilocaeaHM148197 oleagina C. tenuissimum GU214634 D. lagerstroemiae AY725526 P. pseudoeucalyptorum HM148094 C. funiculosum AF338400 Spilocaea oleagina 57 JQ948454 93 AF338401 Spilocaea oleagina C. sphaerospermum complex 0.005 GU269660 P. balsaminae HM148095 C. gamsianum JN906978 EU035438 C. aphidis Venturia tremulae 51 GU214633 D. hilliana JF951148 D. queenslandica C. phormii JQ948448 73 GU269695 P. dianellae HM148096 C. globisporum AF338403 Spilocaea93 AF338400 oleagina Spilocaea oleagina KF793785C. sphaerospermum Venturia saliciperda complex C. pyricola JQ948445 98 DQ780374 C. salinae GU269723 JF951148 P. humuli D. queenslandica 0.02 HM148097 C. hillianum AF338403 Spilocaea oleagina 82 HQ599605 D. xanthorrhoeae C. salicis JQ948478 EU03546 Venturia DQ780374 pyrina C. salinae GU269751 P. nephrolepidis HM148101 C. inversicolor 86 AF338402 Spilocaea oleagina 82 HQ599605 D. 63xanthorrhoeae C. acerbum JQ948459 86 AF338402 Spilocaea oleagina GU269787 P. profusa HM148110 C. iranicum KF156040 Venturia inaequalis JX06987538 D. agapanthi JX069875 D. agapanthi C. rhombiforme JQ948457 AF338399 Spilocaea oleagina AF338399 Spilocaea oleagina AUR38 121 (125632) GU269792 P. crocea HM148111 C. licheniphilum 63 0.005 DQ303077 P. natalensis D HM148115 C. lycoperdinum EU035438 Venturia tremulae 0.005 AUR 2 (8) 65 EU035438 Venturia tremulae GU269780 P. natalensis HM148117 C. myrtacearum 0.005 0.005 98 KF793785 Venturia saliciperda FJ1509020.02 A. GU269730 pullulans P. kiggelariae var. pullulans HM148120 C. paracladosporioides KF793785 Venturia saliciperda FJ150908 A. pullulans GU269734 var. pullulans P. longispora HM148121 C. perangustum 98 EU03546 Venturia pyrina 0.02 COLL 4 (37) FJ150900 A.pullulans71 var. pullulans GU269747 P. musae JF499837 C. phaenocomae EU03546 Venturia pyrina KF156040 Venturia inaequalis Aureobasidium spp. AF362054 P. cordiana HM148153 C. phyllactiniicola COLL 5 (16) F 28 AUR 1 (125632) GQ852770 P. tereticornis AUR 3 (49) HM148154 C. phyllophilum 68 JQ005175 C. karstii KF156040 Venturia inaequalis AUR 2 (8) D GU269650 P. acericola FJ150875 A. pullulans var. namibiae FJ936160 C. piniponderosae JQ005221 C. phyllanthi 95 JQ005222 C. annellatum 0.005 AUR FJ150902 1 (125632) A. pullulans var. pullulans GU269656 P. assamensis FJ150886 A. pullulans var. melanogenum HM148158 C. pseudocladosporioides GU269663 P. callicarpae HM148193 C. rectoides Other C. boninense s.l. species FJ150893 A. pullulans var. subglaciale 66 FJ150908 A. pullulans var. pullulans 80 GU269770D P. rhapisicola HM148195 C. scabrellum AUR 2 (8) JQ005236 C. dacrycarpi JQ324983 P. xanthocercidis EF679354 C. silenes FJ150900 A.pullulans var. pullulans JQ005237 C. constrictum HM148196 C. subuliforme 0.005 FJ150902 A. pullulans var.AUR 3pullulans (49) Other Pseudocercospora species 86 JQ005238 C. constrictum HM148224 C. varians 0.005 5 JQ005152 C. gloeosporioides 95 FJ150875 A. pullulans var. namibiae HM148226 C. verrucocladosporioides FJ150908 A. pullulans var. pullulans Other Pseudocercospora species FJ150886 A. pullulans var. melanogenum HM148230 C. xylophilum 0.005 FJ150900 A.pullulans FJ150893 var. A. pullulans pullulans var. subglaciale 88 CLA 3 (7) 80 Cladosporium spp. JF770450 C. grevilleae AUR 3 (49) 0.01 DQ780379 C. langeronii 63 DEV 1 (5976) 87 DQ780386 C. psychrotolerans G DEV 2 (18) FJ150875 A. pullulans var. namibiae 98 DEV 3 (11) 0.005 95 SPIL (75) C. herbarum complex 51 DEV 4 (4) KF766166 Spilocaea oleagina 57 FJ150886 A. pullulans var. melanogenum B HQ599602 D. fraseriae EU035435 Fusicladium phillyreae HM148067 C. colocasiae JX294932 D. ficus 65 EU035434 Spilocaea oleagina FJ150893 A. pullulans var. subglaciale 83 HM148197 C. tenuissimum GU214634 D. lagerstroemiae 80 AF338401 Spilocaea oleagina JN906978 C. aphidis 51 GU214633 D. hilliana AF338400 Spilocaea oleagina 93 C. sphaerospermum complex JF951148 D. queenslandica AF338403 Spilocaea oleagina DQ780374 C. salinae HQ599605 D. xanthorrhoeae AF338402 Spilocaea oleagina 82 86 JX069875 D. agapanthi AF338399 Spilocaea oleagina 38 0.005 EU035438 Venturia tremulae 0.005 98 KF793785 Venturia saliciperda 0.02 EU03546 Venturia pyrina KF156040 Venturia inaequalis AUR 1 (125632) AUR 2 (8) D 0.005 FJ150902 A. pullulans var. pullulans FJ150908 A. pullulans var. pullulans FJ150900 A.pullulans var. pullulans AUR 3 (49) 95 FJ150875 A. pullulans var. namibiae FJ150886 A. pullulans var. melanogenum 80 FJ150893 A. pullulans var. subglaciale

0.005 Sampling: May, June, October, December

Colletotrichum spp. specific primers

Analysis of sequences and DNA Sanger phylogentic analyses along Amplification Cloning extraction sequencing with validate panels of reference databases C. gloeosporioides sensu lato C. boninense sensu lato

C. gloeosporioides s.s. C. karstii? C. godetiae

Colletotrichum sp.

C. scutatum s.s.

C. acutatum sensu lato C. kahawae PLoS ONE 11(8): e0160470.

Investigation of disease aetiology

Tarocco Scirè Lane Late Tangelo

Florida: S. H. Futch and L. W. Timmer (https://edis.ifas.ufl.edu/hs263) Sicily: Southern Italy The etiology of greasy spot is still controversial although Mycosphaerella citri (anamorph Zasmidium citri-griseum) is considered the causal agent. Ref: Mondal and Timmer 2006. Plant disease 90: 532-538 Mycosphaerella spp. was detected in all investigated samples: there were no significant differences between symptomatic and asymptomatic sample 90 MID19 55(17299) 48 GU214691 Ramularia brunnea CPC4903 AF173312 Ramularia grevilleana CPC656 86 EU019284.2 Ramularia pratensis var. pratensis CPC11294 Mycosphaerella citri AY490763 Ramularia endophylla CBS113265 GU214693 Ramularia sp. CPC11297 does not seem to be 18 40 GU214689 Ramularia acroptili CBS120252 100 FJ839626 Zasmidium anthuriicola CBS118742 100 KF901779 Zasmidium citri-griseum CPC13467 the causal agent of KF901739 Zasmidium indonesianum CPC15300 Zasmidium 20 52 DQ303023 Zasmidium pseudoparkii CBS110999 greasy spot in EU041798 Ramichloridium cerophilum CBS103.59 62 KP896043 Zasmidium fructicola ZJUM9 100 84 KP896056 Zasmidium fructigenum ZJUM36 southern Italy 18 GU214695 Ramularia rufomaculans CPC10852 MID19 1767(189) Mycosphaerellaceae 86 AF362068 Mycosphaerella graminicola CBS110744 GU214670 Phloeospora maculans CBS115123 GU214668 Passalora sp. CPC12319 16 48 AF222831 Mycovellosiella bellynckii CBS150.49 None of the detected 88 MID7 4593(252) AF173314 Mycosphaerella africana CBS116154 sequences clustered with 70 AF173300 Mycosphaerella keniensis CBS111001 AY725545 Mycosphaerella ellipsoidea CBS110843 reference sequences of M. citri MID10 3230(352) 86 GU214703 Thedgonia-like genus CPC12304 AY489285 Septoria obesa CBS354.58 MID10 154(122) 54 GU214697 Septoria convolvuli CBS102325 86 AY489272 Septoria senecionis CBS102366 AY152574 Septoria apiicola CBS400.54 DQ303085 Pseudocercospora sp. CPC11592 MID19 286(436) 42 AY725520 Dissoconium aciculare CBS204.89 30 AY725550 Dissoconium dekkeri CBS111169 Dissoconiaceae 100 MID10 3538(16) 96 68 AY725535 Dissoconium commune CBS110747

0.05 Role of key insects as local vectors of plant pathogens Bark beetle

Ambrosia beetle

Role of key insects as international vectors of plant pathogens

15 Italian harbours Bark beetle

Ambrosia beetle Diffusion and diversity of Phytophthora species

Fruit of pomelo (Citrus grandis) with symptoms of brown rot caused by mixed infections of P. sp. prodigiosa and P. sp. mekongensis from Vietnam (Puglisi et al., 2017: PLoS ONE 12(2): e0172085).

Analysis of sequences and DNA Sanger phylogentic analyses along Amplification Cloning extraction sequencing with validated panels of reference databases

Molecular analysis of Phytophthora diversity in nursery-grown ornamental and fruit plants

Prigigallo et al., 2015: Plant Pathology 64, 1308– 1319 Nursery TP Nursery BL 100% 100% Prigigallo et P. cinnamomi P. niederhauserii-like al., 2016. 90% P. palmivora-like 90% P. palmivora Phytopatholo 80% P. lateralis 80% P. citrophthora Phytophthora sp. P. meadii-like 70% 70% gy 106: 305- P. ramorum P. cryptogea 313. 60% P. palmivora 60% P. citricola 50% P. niederhauserii 50% Phytophthora sp. P. cinnamomi-like P. cambivora 40% 40% P. cryptogea P. niederhauserii

30% P. nicotianae 30% P. meadii Relative abundance (%) abundance Relative Relative abundance (%) abundance Relative 20% 20% P. nicotianae 10% 10% 0% 0% 454 Sanger 454 Sanger Nursery PV Nursery ZZ 100% 100% P. niederhauserii Phytophthora sp. 90% P. pseudosyringae 90% P. palmivora 80% P. palmivora 80% P. megasperma P. cryptogea 70% P. cryptogea 70% P. lateralis P. pseudosyringae-like 60% 60% P. nicotianae P. pseudosyringae 50% 50% P. lateralis P. cinnamomi-like 40% 40% P. taxon Pgchlamydo

30% 30% Relative abundance (%) abundance Relative Relative abundance (%) abundance Relative 20% 20% 10% 10% 0% 0% 454 Sanger 454 Sanger Nursery VM Nursery CP 100% 100% P. meadii P. parvispora 90% P. nicotianae 90% P. palmivora 80% 80% P. meadii 70% 70% P. lateralis 60% 60% P. niederhauserii P. nicotianae 50% 50% P. cryptogea 40% 40% P. citrophthora

30% 30% Relative abundance (%) abundance Relative Relative abundance (%) abundance Relative 20% 20% 10% 10% 0% 0% 454 Sanger 454 Sanger Nursery PG Nursery ST 100% 100% P. palmivora P. cactorum 90% P. cryptogea 90% P. nicotianae 80% P. citrophthora 80% P. cryptogea P. niederhauserii 70% P. citricola 70% P. cambivora 60% 60% 50% 50% 40% 40%

30% 30% Relative abundance (%) abundance Relative Relative abundance (%) abundance Relative 20% 20% 10% 10% 0% 0% 454 Sanger 454 Sanger Development and validation of qPCR detection methods

Venturia oleaginea : causal agent of peacock leaf spot in olive

Bitroph pathogen: pure culture isolates are not available.

Sanger Analysis of sequences and Collection of Sequencing DNA Amplification phylogentic analyses along symptomatic extraction (Ve1F-Ve1R) with validate panels of leaves MiSeq reference databases Sequencing Results of metabarcoding analyses:

A region shared by all sequences of Ventuaria oleaginea was identify and used to develop specific primers and a TaqMan probe

Specific primers and probe

Venturia oleaginea is the only fungus of the family Venturiaceae associated to the olive phyllosphere Future perspectives All currently DNA barcode studies Metabarcoding analyses indicate the need for more based on the ITS region alternative genes with different should be integrated with level of discrimination to accurately identify fungal and new DNA barcode regions oomycete taxa

An increased lenght of the ITS Gene 1 Gene 2 Gene x amplicons may help (third- generation HTS) but may not be ITS rDNA: primary barcode resolutive Other genes: secondary DNA barcodes Future perspectives New target regions for metabarcoding analyses: characterized regions utilized in barcode analyses are the best candidate because DNA databases are already available New primers may be needed Future perspectives

Other appropriate genomic regions for amplicon metagenocims approches may be identified comparing available fungal genomes

Primer F Primer R

Conserved Conserved Variable region gene gene

Appropriate lenght Summary of major challenges

Specificity (amplification Simplified from non-target bioinformatics organisms) platforms Creation and or Identification of Design and implementation new target validation of of validated regions Sanger appropriate reference sequencing primers databases Lenght of the Coverage of amplicons primers

Second- Third- Universal primers Primers with different levels generation generation covering all fungal of specificity: genus, family, HTS HTS taxa order, class, phylum etc. Acknowledgements

Prot. 20172TZHYX: RBFR10PZ4N: Santina Samir Michael Metagenomic strategies to A gnotobiotic-based approach Gaetano PON03PE_00090_1 assess genetic diversity in to unravel the role of the PON03PE_00090_2 soil-borne Phytophthora plant microbiome and PON03PE_00090_3 species develop synthetic PON03PE00026R communities increasing plant growth and stress tolerance

Gianni Silvia David

Antonino

Saveria Ahmed Marisabel Giulia Maria Antonio Sonia