New insight into Black Pod Disease of Cacao
Dr. Bryan Bailey Dr. Shahin S. Ali, Jonathan Shao, Mary D. Strem and Dr. Lyndel Meinhardt SPCL/BARC Beltsville, MD
Thanks to all our collaborators
Ishmael Brett Tyler Mark Guiltinan Amoako-Attah Black pod of cacao It is one of the most economically serious problems for all cacao growing areas. Annual losses due to black pod rot may range from 30-90% of the crop
Phytophthora “the plant-destroyer” Oomycetes (water molds)
Phylogenetic tree illustrating the approximate relationship between oomycetes and fungi Source: Nature Reviews Microbiology 3, 47-58. Source: 2002 Oomycetes. The Plant Health Instructor. • The exact taxonomic designation of the genus Phytophthora remains a subject of intense discussion (Bret Tyler, Oregon State University).
• Kingdom: Stramenopila (sometimes spelled Straminopila)/Chromista; Phylum Oomycota/Heterokontophyta; Class Peronosporomycetidae/Oomycetes; Order Peronosporales; Family Peronosporaceae; Genus Phytophthora (Dick, 2001; Thines, 2014). Global distribution of Cacao Diseases
SPCL Phytopthora megakarya
Phytopthora palmivora
Phytophthora citrophthora
Phytophthora capsici/tropicalis We know very little about P. citrophthora and P. capsici. The majority of the literature is older and hard to access. Although these species were considered very important in South and Central America in the past, new isolates obtained from Brazil, Trinidad, and Costa Rica were all P. palmivora. To add to the confusion, P. capsici isolates from tropical trees have largely been re-classified as a new species, P. tropicalis. The collection of these species from cacao and their careful genetic analysis is warranted. Any results should be published in a readily assessable format.
That other Phytophthora species are occasionally isolated from cacao should not be a surprise. What is missing with rare species is some measure of their true potential for causing disease in cacao. P. megakarya
P. megakarya was first identified as a species in 1979. Likely present in Nigeria prior to 1960 and even earlier, In recent years, P. megakarya seems to have displaced P. palmivora from Cameroon and Nigeria. Pod losses to P. megakarya can be 60-100% in the infected areas of Ghana. How to differentiate between P. palmivora and P. megakarya ?
P. megakarya P. palmivora Phytophthora Phytophthora megakarya palmivora
A Internal Transcribed Spacer 2 Phytophthora capsici G CTCGAAAAG CGTGGTGTTGCTGGTTGTGGAGGCTGCCTGCGTGGCCAGT 175 Phytophthora citrophthora G CTCGAAAAG CGTGGTGTTGCTGGTTGTGGAGGCTGCCTGCGTGGCCAGT 180 Phytophthora palmivora GCTCC AAAAGCGTGGCGTTGCTGATTGTGGAGGCTGCTTGCGTAGCCAGT 181 Phytophthora megakarya GCTCGAAAAG TAAAGC-TTGCTTGTTGTGGAGGCTGCTTGTGTAACCAGT 179 **** ***** .:.* ***** .************* ** **..*****
Phytophthora capsici C - GGCGACCGGTTTGTCTGCTGCGGCGTTTAATGGAGGAGTGTTCGATTC 224 Phytophthora citrophthora C - GGCGACCGGTTTGTCTGCTGCGGCGTTTAATGGAGGAGTGTTCGATTC 229 Phytophthora palmivora CTGGCGACCAGTTTGTCTGCTGTGGCG -TTAATGGAGGAGTGTTCGATTC 229 Phytophthora megakarya C - GGCGACTAGTTTGTCTGCTGTGGCG -TTAATGGAGGAGTGTTCGATTC 227 * ****** .************ **** **********************
Phytophthora capsici GCGGTATGGTTGGCTTCGGCTGAACAGGCGCTTATTG TATGCTTTTCCTG 274 Phytophthora citrophthora GCGGTATGGTTGGCTTCGGCTGAACAGGCGCTTATTG TATGCTTTTCCTG 278 Phytophthora palmivora GCGGTATGGTTGGCTTCGGCTGAACAGACGCTTATTGAATATTTC TTCAG 279 Phytophthora megakarya GCGGTATGATTGGCTTCGGCTGAACAGAA GCTTATTGGGCGTTTTTCCTG 277 ********.******************..******** . . ** * *:*
Phytophthora capsici CTGTGG CGTGATGGGCTGGTGAACCGTAG 303 Phytophthora citrophthora CTGTGG CGTGATGGGCTGGTGAACCGTAG 307 Phytophthora palmivora CTGTGG TGGTATG-ATTGGTGAACCGTAG 307 Phytophthora megakarya CTATGGCGGTATGAAGTAGTGAACCGTAG 306 **.*** * *** . *.*********** P. megakarya P. plamivora
Annealing temperature Annealing
Multiple sequence alignment of ITS region for 4 different Phytophthora spp. responsible for black pod of cacao and derivation of species specific PCR primers. Molecular isolates West Africa, collected phylogenetic Central from and infected analysis South America cacao of Phytophthora pods from
P. Palmivora Cld. 4
Ivory Cost Geographical megakarya causing 25,26 1324, 411 100 WR 24 415 420 130 black 254 113 10 236 14 133 50 BAR 1327 256 and distributions pod AR 256 246 233 251 61 204 Phytophthora Phytophthora 243 255 205 of CR 1333 P . 1340 215 cacao 354 240 352 360 1331,34 1330 1341 palmivora 1349 NR ER 344 1332 1334,35 349 1346, 48 of palmivora megakarya 340 in 1348 Phytophthora . Ghana GA W 112 101 100 106 105 VR S N isolates 93 95 E ,36 12,13,21,23 1303,07,11, ,17,19,37 06,09,14,16 1301,02,05, 1315,18,0,22
Togo A 3 hpi 3 hpi ------IC Ca Gh Gh Gh Gh Gh Gh Gh Gh Gh Gh Gh Gh Gh Gh AR61 VR105 VR100 CR233 CR255 WR411 WR420 ER1349 ER1334 ER1331 VR1306 A VR1315 VR1307 ZTHO145 Control BAR1324 SBR112.9
B 100 80 60 5 hpi 5 hpi 40 20
Necrotic area (%) area Necrotic 0 AR61 - VR105 VR100 CR255 CR233 WR411 WR420 - - - - Control - - VR1315 VR1307 ER1334 ER1331 VR1306 ER1349 ------Gh BAR1324 ZTHO145 SBR112.9 - - Gh Gh Gh Gh - Gh Gh 100 µm Gh Gh Gh Gh Gh Gh IC Ca Gh Phytophthora megakarya Phytophthora palmivora
Phytophthora megakarya Phytophthora palmivora B
Difference in virulence, zoospore germ tube length, and infection structures between Phytophthora megakarya and P. palmivora during early infection of cacao pod. C Note isolates within each species vary in agressiveness but P. megakarya is more consistently aggressive.
P. megakarya produces appresoria much more often than P. palmivora. Both species penetrate through stomata. Hours post-inoculation P. megakarya Gh-ER1334 P. palmivora Gh-ER1349 Is megakarya really aggressive?
A Gh-VR1315
Ca-ZTHO145
megakarya Gh-ER1334 Phytophthora
IC-SBR112.9
Gh-ER1349 palmivora
Phytophthora Gh-VR100
Control 1 cm
1 2 3 4 Days post-inoculation B Gh-VR1315 Ca-ZTHO145 Gh-ER1334 IC-SBR112.9 Gh-ER1349 Gh-VR100 Control 0 1 2 3 4 5 6 AUDPC (cm2)
Differential disease progressions under wounded system in response to P. megakarya and P. palmivora infection of cacao pod husk. Differential disease progression responses to P. megakarya and P. palmivora infection of wounded and attached cacao pods of tolerant clone. Fluorescent tagging of P. palmivora and P. megakarya
Appressorium Plant cell wall Zoospore cyst
Intercellular hyphae
Howard Judelson’s lab: Fungal Biology 115 (2011) 882-890. Intercellular Haustoria hyphae
Leaf tissue infected with GFP tagged P. palmivora
Leaf tissue infected with mCherry tagged P. palmivora Leaf tissue infected with GFP tagged P. megakarya Phylogenetic difference between P. palmivora and P. megakarya
Genome sequencing P. megakarya isolate ZTHO120 P. palmivora isolate SBR112.9
Blair et al (2008) FGB, 45-266-77 Assembly and annotation statistics
Comparison of P. megakarya and P. palmivora gnome P. megakarya P. palmivora
Total contig length 101,182,312 107,423,419 Contig numbers 27,143 28,632 CEGMA Completeness (%) 94.35 96.37 GC content 48.92% 48.91% N50 contig length 6,902 6,456 Scaffold number 24,070 24,815 Gene number 41,992 44,305 Total gene length 42,723,254 45,995,141 Average gene length 969.78 1038.15 Hypothetical protein with 24,099 20,785 unknown functions Genes with no similarity to 788 589 proteins in Nr database Genome size
Total number of genes
250 17,797
200 25,852 Estimation of genome size based on K-mer 44,305 25,493 41,992 statistics 150 P. megakarya P. palmivora 100 16,988 Ave. read length 90 90 14,451 50
K-mer length 15 15 (Mb) size Genome Coverage depth 33.41 33.25 0 K-mer number 4,388,028,112 5,057,947,212 K-mer depth 28.01 28.03 Genome size (Mb) 156.65 180.44 P. sojae Contig 64.58% 59.53% * P infestans representation # P. ramorum
P. palmivora * P. megakarya P. *Science 2006, 313(5791):1261-6 #Nature 2009, 461(7262):393-8 Ploidy level ?
Phylogeny of some other house-keeping genes
Ppal Cat _ a TCACACATGGTTCCGGGCATTGAG CCGTCTCCGGACAAGATGCTTCAAGGTCGTTTGTTC 1020 Ppal Cat _b TCACACATGGTTCCGGGCATTGAGC CGTCCCCAGACAAAATGCTTCAAGGTCGTTTGTTC 1020 ***************************** **.*****.*********************
Ppal Cat _ a TCCTATCCAGATACGCAGCGCCACCGTCTGGGTGCCAACTACAATCAGATCCCAGTGAAT 1080 Ppal Cat _b TCCTATCCGGATACGCAGCGCCACCGTTTGGGTGCCAACTACAATCAGATCCCAGTGAAT 1080 ********.****************** ********************************
Ppal Cat _ a CGACCGCTCAAGGTGCCACAGACGTACCAGCGTGACGGCTTCATGGCTATCAATGGCAAC 1140 Ppal Cat _b CGACCGCTCA AGGTGCCACAGACATACCAGCGTGATGGCTTCATAGCTATCAATGGCAAC 1140 ***********************.*********** ********.***************
Ppal Cat _ a ATGCACGACACGCCAAACTACTTTCC GAACAGCAAGAATGGCCCGGCTGAGGACACAACA 1200 Ppal Cat _b ATGCACGACGCGCCAAACTACTTT CCAAACAGTAAGAATGGTCCGGCTGAGGACACAACA 1200 Phylogeny of all Histone deacetylases from *********.****************.***** ******** ****************** P. megakarya (10 proteins) & P. palmivora (16 proteins). Different P. palmivora isolates Comparison of genes coding for transporters
Gene products P. megakarya P. palmivora No. of genes No. of genes in for P. sojai* Diatom* No. of genes No. of genes
ABC super-family 450 894 134 63 PDR 62 113 67 9 MRP 54 84 23 6 MFS transporters 90 118 n.d n.d Voltage-gated ion channels 46 94 n.d n.d
Mitochondrial carriers 57 95 n.d n.d
Ankyrin family 81 75 n.d n.d Voltage-gated K+ channels 25 49 n.d n.d
Sugar transporter 13 37 n.d n.d
*Science 2006, 313(5791):1261-6 Comparison of genes coding for hydrolases
Gene products P. megakarya P. palmivora No. of genes for No. of genes in P. sojai* Diatom* No. of genes No. of genes
Proteases, all 409 499 282 314 Serine proteases 182 208 119 123 Aspartic proteases 49 37 12 n.d Cysteine proteases 78 73 57 63 Glycosyl Hydrolases, all 291 423 125 n.d Chitinases 11 8 5 49 Cutinases 9 7 16 0 Pectinases 59 103 62 0 Pectinesterase 17 21 Lipases 62 105 171 n.d
*Science 2006, 313(5791):1261-6. Phylogeny of all cystein proteases from P. Phylogeny of all aspartic proteases from P. megakarya (78 proteins) & P. palmivora (73 megakarya (49 proteins) & P. palmivora proteins). (37 proteins). Comparison of genes eliciting necrosis
Gene products P. megakarya P. palmivora No. of genes No. of genes in for P. sojai* Diatom* No. of genes No. of genes
Protein toxins NPP family 109 77 29 0 PcF family 6 3 19 0 Crn family 152 139 40 0
Phylogeny of all phytotoxic protein PcF from P. megakarya (6 protein) & P. palmivora (3 proteins). Phylogeny of all NPP family proteins from P. megakarya (109 proteins) & P. palmivora (77 proteins). Comparison of genes coding for effectors
Gene products P. megakarya P. Palmivora No. of genes No. of genes in for P. sorji* Diatom* No. of genes No. of genes
Elicitins 52 56 57 0 Avh (RxLR) family 999 873 350 0
Build two HMM models from candidates: 1. RXLR motif and 10 amino acids on the left side; 2. The complete RXLR-dEER domain Putative RxLRs
Use HMMER to search all six-frame translations from the complete genome sequences (1E-5) P. megakarya 281
P. Palmivora 242 Get all RxLR genes
PSI-Blast (1E-5) Phytophthora secrete RxLR effectors to manipulate plant defense.
Domain organization of four Phytophthora cytoplasmic RxLR effectors
A hypothetical model for RxLR effector secretion and delivery into host cells
Source: Current Opinion in Microbiology 2007 (10) 332-8. Comparison of Retroelements Gene products P. megakarya P. palmivora
No. of genes No. of genes Transposons /retrotransposons 301 215 Transposases 226 164 Reverse transcriptases 2372 1320 Polyprotein 1108 2708 Copia protein 45 25 Eukaryotic/viral aspartic protease 292 28
bp
1/158
21/133 scaffold /
42/299
22/215 megakarya palmivora . . P P 32/35
Repeat driven genome expansion of P. megakarya In conclusion
Both P. megakarya and P. palmivora have the largest number of genes among the 5 Phytophthora genomes sequenced recently. P. palmivora has doubled its ploidy level down the evolutionary path. Retroelements has a major role in the genome expansion of P. megakarya. Higher number of effectors in P. megakarya can be attributed to retroelements that gave it a pathogenic advantage over P. palmivora. Shahin S. Ali1, Jonathan Shao1, Ishmael Amoako-Attah2, Rebecca A. Bailey1, Martha Schmidt1, Mary D. Strem1, Danyu Shen8, Surendra Surujdeo-Maharaj3, Cristiano Villela Dias4, Erica Goss5, B.A. Didier Begoude6, G. Martijn ten Hoopen6, Jean-Philippe Marelli4, Wilberth Phillips-Mora7, Brett Tyler9, Mark Guiltinan10, Lyndel Meinhardt1 and B. A. Bailey
1Sustainable Perennial Crops Laboratory, USDA/ARS, Beltsville, Maryland 2Cocoa Research Institute of Ghana, Akim New-Tafo, Ghana. 3Cocoa Research Centre, the University of the West Indies, St. Augustine, Trinidad and Tobago. 4Mars Center for Cocoa Science-Cp 55 Itajuipe Ba, Brazil Cep 45630-000. 5Plant Pathology Department, University of Florida, Gainesville, FL 32611-0680. 6Regional Laboratory for Biological and Applied Microbiology, IRAD, B.P. 2067, Yaoundé, Cameroon. 7Department of Agriculture and Agroforestry, CATIE, Turrialba 7170, Costa Rica. 8College of Plant Protection, Nanjing Agricultural University, Nanjing, 210095, China. 9Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR, USA. 10Penn State University, PA, USA