Complete sequence and genome characterization of miscanthus virus M, a new betaflexivirus from Miscanthus sp.

A novel betaflexivirus, tentatively named "miscanthus virus M" (MiVM), was isolated from Miscanthus sp. The complete genome of MiVM is 7,388 nt in length (excluding the poly(A) tail). It contains five open reading frames and has a genome organization similar to those of members of the families Alphaflexiviridae and Betaflexiviridae (subfamily Quinvirinae). The amino acid sequences of both the replicase and coat protein shared less than 45% identity with the corresponding sequences of members of either family. Phylogenetic analysis confirmed that MiVM belongs to the family Betaflexiviridae and subfamily Quinvirinae but it was too distantly related to be included in any currently recognized genus in this family. We therefore propose that miscanthus virus M represents a new species and a new genus in the family Betaflexiviridae.

Complete genome characterization of cacao leafroll virus, a newly described cacao-infecting polerovirus.

The complete genome sequence of cacao leafroll virus (CaLRV; family Solemoviridae, genus Polerovirus) was determined by high-throughput sequencing of total RNA isolated from symptomatic cacao Theobroma cacao L. plants (n = 4). The CaLRV genome sequences ranged from 5,976 to 5,997 nucleotides (nt) in length and contained seven open reading frames (ORFs). Nucleotide and amino acid (aa) sequence comparisons showed that, among selected well-characterized poleroviruses, the CaLRV genome shared the highest nt sequence identity of 62% with that of potato leafroll virus (PLRV, NC_076505). A comparison of the predicted aa sequence of the CaLRV coat protein indicated that cotton leafroll dwarf virus (CLRDV, NC_014545) and melon aphid-borne yellows virus (MABYV, NC_010809) were the closest relatives, sharing 57% aa sequence identity. Bayesian phylogenetic analysis based on complete genome sequences showed that CaLRV grouped with well-characterized poleroviruses that cause diseases of cereal and vegetable crops. During the course of publishing this work, the nearly complete genome sequence of a member of the same polerovirus species, referred to as "cacao polerovirus" (OR605721), with which CaLRV shares 99% nt sequence identity, was reported.

Coffee Fruit Rot: The Previously Unrecognized Role of Fusarium and Its Interactions with the Coffee Berry Borer (Hypothenemus hampei).

Coffee fruit rot (CFR) is a well-known disease worldwide, mainly caused by Colletotrichum spp., the most important species being C. kahawae subsp. kahawae. In Puerto Rico, Colletotrichum spp. were identified as pathogens of coffee fruits. The coffee berry borer (CBB) was shown to be a dispersal agent of these fungi, and interaction of Fusarium with Colletotrichum affecting coffee fruits was suggested. In this study, we demonstrated that Fusarium spp. also cause CFR in Puerto Rico. Fusarium spp. are part of the CBB mycobiota, and this insect is responsible for spreading the pathogens in coffee fields. We identified nine Fusarium spp. (F. nirenbergiae, F. bostrycoides, F. crassum, F. hengyangense, F. solani-melongenae, F. pseudocircinatum, F. meridionale, F. concolor, and F. lateritium) belonging to six Fusarium species complexes isolated from CBBs and from rotten coffee fruits. Pathogenicity tests showed that F. bostrycoides, F. lateritium, F. nirenbergiae, F. solani-melongenae, and F. pseudocircinatum were pathogens causing CFR on green coffee fruits. F. bostrycoides was the predominant species isolated from the CBB mycobiota and coffee fruits with symptoms of CFR, suggesting a close relationship between F. bostrycoides and the CBB. To our knowledge, this is the first report of F. bostrycoides, F. solani-melongenae, F. pseudocircinatum, and F. nirenbergiae causing CFR worldwide and the first report of F. lateritium causing CFR in Puerto Rico. Understanding the CFR disease complex and how the CBB contributes to dispersing different Fusarium spp. on coffee farms is important to implement disease management practices in Puerto Rico and in other coffee-producing countries.

First Report of Diaporthe tulliensis Causing Necrotic Spots and Leaf Blight on Rambutan in Puerto Rico.

Rambutan (Nepehelium lappaceum) is a tropical exotic fruit belonging to the Sapindaceae family. Several pathogens have been identified in rambutan causing different diseases on fruits, inflorescences, and branches (Serrato-Diaz et al., 2015, 2017, 2020) but few on leaves. From 2015 to 2021, a disease survey was conducted in one greenhouse in Mayaguez, Puerto Rico and experimental rambutan field orchards of the USDA-ARS Tropical Agriculture Research Station located at Isabela, Corozal, Santa Isabel, and Adjuntas, Puerto Rico (Latitude: 18°12'28"N, 18°34'10'' N, 18°00'47''N, 18°16'35''N and Longitude: 67°08'17"W, 66°31'74'' W, 66°38'98''W, 66°72'32''W, respectively). Varieties Benjai, Gula Batu, Jitlee, R-134, R-156Y, R-162, R-167 and Rongren were sampled. Necrotic spots and leaf blight were commonly observed with a disease incidence of 80%. Diseased leaves showed necrosis starting from the apex and spreading through the lamina. Ten diseased leaves were collected from each location and sections of symptomatic tissue (5mm2) were disinfected and plated on potato dextrose agar (PDA) and oatmeal agar (OA). Two representative isolates of Diaporthe tulliensis, A3 and A4, were obtained, purified, and identified morphologically and by PCR amplifications of three nuclear genes of the Internal Transcribed Spacer ITS1-5.8S-ITS2 region of the ribosomal DNA primers ITS5/ITS4, portions of the ß tubulin (BT) primers Bt2a/Bt2b and the translation elongation factor 1-α (TEF1-α) primers EF1728F/EF1986R. On PDA and OA colonies of isolates A3 and A4 were initially white and flat with sparse mycelia that turned yellowish-white to grey with age. Pycnidia were black with cream to pale yellow conidial droplets that exuded from ostioles. Hyaline, unicellular alpha conidia were oval to cylindrical, rounded at apex and obconically truncate at base. Alpha conidia (n = 50) for isolates of D. tulliensis were 4.9 to 5.9 µm long by 2.2 - 2.5 µm wide. DNA sequences of the ITS region and partial sequences of TEF1-α and BT genes were compared by BLASTN with Diaporthe sequences deposited in GenBank. ITS, BT and EF1-α sequences of isolates A3 and A4 (OP219651 and OP161553 for ITS region; OP222137 and OP168832 for TEF1-α; OP222136 and OP168831 for BT, respectively) were grouped to the holotype BRIP 62248a (Bootstrap BS=100) of Diaporthe tulliensis R.G. Shivas, Vawdrey & Y.P. Tan. Pathogenicity tests were conducted on six of six-months-old rambutan seedlings of R-167 variety. Three unwounded healthy non-detached leaves were inoculated per isolate with one 5mm mycelial disk from pure cultures grown on PDA. Rambutan seedlings were kept in a humid chamber using plastic bags for 8 days under greenhouse conditions. Two of six seedlings were used as controls and inoculated with PDA disks only. Eight and 14 days after inoculation (DAI), D. tulliensis isolates caused necrotic spots and leaf blight, on leaves. Diseased leaves turned from light to dark brown starting from the apex and spreading through the lamina with necrotic lesions ranging in size from 5 - 10 mm. Untreated controls showed no symptoms, and no fungi were re-isolated from tissue. D. tulliensis was re-isolated from diseased leaves, fulfilling Koch's postulates. D. tulliensis has been reported in Taiwan causing Diaporthe leaf spot in Boston Ivy (Huang, C. et al., 2021) and Bodhi trees (Li, K.Y. et al., 2022), as well as Jasmin stem canker (Ching Hsu, C. et al., 2022). It has been reported as causing leaf blight of coffee (Gong, J. L., et al., 2019), kiwifruit stem canker in China (Bai et al., 2017), and most recently causing cacao pod rot in Puerto Rico (Serrato-Diaz, L.M. et al., 2022). To our knowledge, this is the first report of Diaporthe tulliensis causing necrotic spots and leaf light on rambutan in Puerto Rico and often associated with a potassium deficiency in many parts of the world. It will be important to establish an adequate and effective control management of this disease in rambutan producing countries worldwide. References and doi hyperlinks: 1. Huang, C. et al. Plant Dis. 105:2718, 2021 https://doi.org/10.1094/PDIS-12-20-2652-PDN 2. Li, K.Y. et al. Plant Dis. 0:ja, 2022 https://doi.org/10.1094/PDIS-01-22-0211-PDN 3. Ching Hsu, C. et al. Plant Dis. 0:ja, 2022 https://doi.org/10.1094/PDIS-09-21-1908-PDN 4. Gong, J. L., et al. Plant Dis. 104:570, 2019 https://doi.org/10.1094/PDIS-09-19-1833-PDN 5. Bai et al. Plant Dis. 101:508, 2017 https://doi.org/10.1094/PDIS-10-16-1445-PDN 6. Serrato-Diaz L.M., et al. 2015. Plant Dis. 99: 1187. https://doi.org/10.1094/PDIS-09-14-0923-PDN 7. Serrato-Diaz L.M. et al. 2017. Plant Dis. 101: 1043. https://doi.org/10.1094/PDIS-11-16-1557-PDN 8. Serrato-Diaz, L.M., et al. 2020. Plant Dis. 104: 105-115. https://doi.org/10.1094/PDIS-02-19-0295-RE 9. Serrato-Diaz, L.M. et al. 2022 Plant Dis. 106: 2530. https://doi.org/10.1094/PDIS-12-21-2634-PDN.

First Report of Diaporthe tulliensis and D. pseudomangiferae causing cacao pod rot in Puerto Rico.

Worldwide cacao pod rot is a devastating disease of Theobroma cacao, infected cacao pods turn necrotic reducing yield up to 30%. From July 2020 to August 2021, a survey was conducted at the USDA-ARS cacao germplasm collection located at Mayaguez, Puerto Rico. Incidence of cacao pod rot was 73.9%, observed in 142 of the 196 accessions sampled. The disease was observed at different stages of pod development (small, green, mature pods, and dry mummified large pods). Diseased tissue from three cacao pods (1 mm2) per each cacao accession was surface disinfested by immersion in 70% ethanol for one minute, rinsed with sterile-distilled-water and plated on potato dextrose agar (PDA) amended with 250 mg/L ampicillin and 60 mg/L streptomycin. After 30 days of incubation at 25°C, seven isolates developing white fast-growing colonies with black-globose pycnidia were observed. All isolates produced hyaline, one-celled, biguttulate, and cylindrical and rounded at the apex α conidia of 5.1 to 7.3 µm × 2.5 to 3.0 µm in size and were identified as Diaporthe spp. (Gomes et al. 2013; Crous et al. 2015). To determine the species identity, seven isolates were sequenced of the internal transcribed spacer (ITS), sections of ß-tubulin (BT) and translation elongation factor 1 alpha (EF1-α) and compared using the BLASTn with Diaporthe spp. type specimens deposited in NCBI GenBank. ITS, BT and EF1-α sequences of Phomocac16, Phomcac17, Phomcac18 and Phomcac21 isolates (GenBank accession nos. OL353698 to OL353701, OL412430 to OL412433, and OL412437 to OL412440 for ITS, BT and EF1-α, respectively) were grouped to the holotype BRIP 62248a (Bootstrap BS=100) of Diaporthe tulliensis R.G. Shivas, Vawdrey & Y.P. Tan. The other three isolates (Phomcac8P1, Phomcac8P3 and Phomcac8P4) were grouped to the ex-type (CBS 101339) of Diaporthe pseudomangiferae R.R. Gomes, Glienke & Crous, ITS, BT and EF1-α sequences of (GenBank accessions nos. OL353702 to OL353704, OL412434 to OL412436, and OL412441 to OL412443, for ITS, BT and EF1-α, respectively). Pathogenicity tests were conducted using isolates Phomocac16, Phomcac17, Phomcac18 and Phomcac21 of D. tulliensis and isolates Phomcac8P1, Phomcac 8P3 and Phomcac8P4 of D. pseudomangiferae on five healthy detached green, yellow and red pods of the following cacao varieties: TARS27, ICS16, ICS1, GS29, UF601, SIAL56, Amelonado, SIAL98, EET94, ICS129 and GNV58. Cacao pods were wounded and inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown on PDA of each isolate and wrapped with parafilm. Untreated controls were inoculated with PDA disks only. Fruits were kept in a humid chamber for 8 days at 25°C. Tests were repeated twice. Eight days after inoculation with D. tulliensis and D. pseudomangiferae, all cacao pods turned dark brown, untreated controls showed no symptoms of pod rot, and no fungi were isolated from tissue. Both species, D. tulliensis and D. pseudomangiferae were reisolated from their respective diseased tissues fulfilling Koch's postulates. Diaporthe tulliensis has been reported from rotted stem ends of cacao pods in Australia (Crous et al. 2015), and D. pseudomangiferae was reported in a shipment of cacao seed pods in California; however, pathogenicity tests were not conducted at either location. In California D. pseudomangiferae is considered a quarantine pathogen with a temporary Q rating (Chitambar 2017). To our knowledge, this is the first report of D. tulliensis and D. pseudomangiferae causing cacao pod rot in Puerto Rico. Knowing the identity and incidence of these new cacao pathogens is the first step for developing specific control measures and potential sources for resistance to cacao pod rot caused by Diaporthe spp. References: Chitambar J. 2017. California Pest Rating for Diaporthe pseudomangiferae R. R. Gomes, C. Glienke & Crous. https://blogs.cdfa.ca.gov/Section3162/?p=3285 Crous P.W. et al. 2015. Persoonia 35:264. https://doi.org/10.3767/003158515X690269 Gomes R.R. et al. 2013. Persoonia 31:1 http://dx.doi.org/10.3767/003158513X666844.

A Previously Undescribed Polerovirus (Solemoviridae) Infecting Theobroma cacao Germplasm.

Cacao Theobroma cacao L. (Malvaceae) is an economically important crop cultivated in tropical climates for the bean from which chocolate and other products are made (Zarrillo et al., 2018). Virus-like symptoms consisting of discoloration, leaf distortion with downward rolling of leaves, and yellow speckling or mottling (Fig. S1), were observed in imported cacao germplasm at the USDA-ARS-SHRS cacao quarantine facilities in the fall of 2020. Total RNA was isolated from leaves collected from four symptomatic plants using silica RNA extraction method (Rott and Jelkmann, 2001). Ribosomal RNA (rRNA)-depleted RNA samples were used for cDNA library construction, followed by high throughput sequencing on Illumina® NovaSeq 6000 platform (Novogene Corp., Sacramento, CA). Quality-filtered, 150-bp paired-ended reads (2,601,293-3,104,474) were assembled de novo using SPAdes v.3.14.1 (Nurk et al., 2013). The contigs (200,799 to 276,851) were queried against the NCBI virus reference sequence (RefSeq) database using the discontiguous megablast algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi?). The resultant contigs (n=1,344) were 150-nt to 1463-nt in length (k-mer coverage from 6.3x to 26,721.7x) and shared their highest nucleotide (nt) identity with potato leafroll virus (PLRV; NC_001747; genus Polerovirus; family Solemoviridae), at 69.1%-72.8%. The contigs pooled from the four samples were assembled into 15 scaffolds. BLASTn analyses of the 15 scaffolds against the RefSeq database indicated the best matches were to thirteen other polerovirus species, with top hits to cereal yellow dwarf virus-RPV (D10206) and pepper vein yellows virus (LC528383), having similarity scores of 66.2% and 100% respectively. The 15 scaffolds matched to the 5' terminal end, ORF1-2, ORF3, ORF4 and ORF3-5 of the different polerovirus genomes. For confirmatory sequencing, total RNA was subjected to reverse transcription using SuperScript IV (Invitrogen, Carlsbad, CA), followed by RT-PCR amplification with general polerovirus primers PoconF/PoconcpR (Xiang et al., 2008) expected to yield an amplicon of ~1,400-bp located at the 3' end of the RNA-dependent, RNA polymerase (RdRp), including the complete coat protein (CP) and movement protein (MP) genes. Amplicons were ligated to pGEM-T Easy vector (Promega, Madison, WI) and sequenced bi-directionally by Sanger sequencing (Eton Bio, Research Triangle Park, NC). BLASTn analysis of the polerovirus-like nt sequences (GenBank accession nos. (ON745771-ON745774) indicated the closest relatives were potato leafroll virus (OK058524) and cucumber aphid-borne yellows virus (FJ460218), at 71% and 73%, respectively. The CP amino acid (aa) sequence shared the greatest similarity to cereal yellow dwarf virus RPV (NP_840023), at 53%, and the MP aa sequence shared the greatest aa similarity to wheat yellow leaf dwarf virus-GPV (YP_003029842), at 38%. These results provide robust support for the association of a previously undescribed polerovirus with symptomatic cacao trees, herein named, cacao leafroll virus (Solemoviridae; Polerovirus). Although Koch's postulates have not been completed to confirm causality, the presence of this virus in cacao germplasm undermine efforts to distribute pathogen-free germplasm and may pose a risk to cacao production in trees established from virus-infected plant material. To our knowledge, this is the first report of a polerovirus infecting cacao trees. All trees of these accessions at the quarantined facility in Miami, FL have been destroyed.

First Report of Neoscytalidium dimidiatum Causing Stem Canker on Dragon fruit (Hylocereus spp.) in Puerto Rico.

Dragon fruit or pitahaya (Hylocereus spp.) is a tropical fruit belonging to the Cactaceae. It is native to Central and South America and commercially grown in the United States in southern California, south Florida and Puerto Rico. During a disease survey from April to June 2020, stem canker was observed in greenhouses and commercial orchards located in Mayaguez and San Sebastian, Puerto Rico with an incidence of 80%. Diseased cladodes (stems) of 1 mm2 tissue sections of 23 pitahaya varieties (NOI-13, NOI-14, NOI-16, N97-15, N97-17, N97-18, N97-20, N97-22, American Beauty, Cosmic Charlie, Halley's comet, Purple Haze, Alice, Bloody Mary, Dark Star, David Bowie, Delight, Makisupa, Red Jaina, Soul Kitchen, Vietnamese Jaina, Neitzel and Lisa) were disinfested with 70% ethanol, rinsed with double distilled water and plated on potato dextrose agar (PDA) amended with 60 mg/L streptomycin. Three isolates (17B-173-T3, 12C-118-T1 and 13B-131-T2) of Neoscytalidium dimidiatum (syn. N. hyalinum) were identified using taxonomic keys (Crous et al., 2006) and sequencing of the internal transcribed spacer (ITS) with primers ITS5 and ITS4 (White et al. 1990) and translation elongation factor 1 alpha (TEF1-α) with primers EF1-728F and EF1-986R (Carbone and Kohn, 1999). Sequences were compared using the BLASTn tool with N. dimidiatum deposited in NCBI GenBank. In PDA, colonies of N. dimidiatum were initially powdery white and turned grayish-black with age. Arthroconidia (n=50) were dark brown, disarticulating, truncate or cylindrical at the base, thick-walled with 0 to 1 septum, averaging 9.1 X 5.5um in length. GenBank accession numbers of N. dimidiatum DNA sequences were MT921260, MT921261 and MT921262 for ITS and MT920898, MT920899 and MT920900 for TEF1-α. Sequences were 99-100% identical with Ex-isotype CBS145.78 accession numbers KF531816 for ITS and KF531795 for TEF1-α. Pathogenicity tests were conducted on 12 healthy dragon fruit plants of 1.5 years old using three non-detached cladodes per plant. Cladodes were inoculated with 5mm mycelial plugs from 8-day-old pure cultures grown on PDA. Three healthy dragon fruit plants were used as controls and were inoculated with PDA plugs only. The experiment was repeated once. Twenty days after inoculations (DAI), isolates of N. dimidiatum caused stem canker on dragon fruit plants. For all isolates, sunken orange spots averaged 3 X 2 mm in length at 8 DAI. Necrotic blotches with chlorotic halos averaged 10 X 15 mm at 14 DAI; stem cankers with water-soaked tissue were observed at 20 DAI, and arthroconidia and black pycnidia on dry stem cankers at 30 DAI. Untreated controls had no symptoms of stem canker, and no fungi were isolated from tissue. Neoscytalidium dimidiatum has been reported to cause stem canker on Hylocereus spp. in China, Florida, Israel, Malaysia and Taiwan (Chuang et al. 2012; Lan et al., 2012; Ezra et al., 2013; Sanahuja et al., 2016). To our knowledge, this is the first report of N. dimidiatum causing stem canker on dragon fruit in Puerto Rico. References: 1. Carbone, I., and Kohn, L. 1999. Mycologia, 91:553. doi:10.2307/3761358 2. Chuang, M. F. et al. 2012. Plant Disease 96: 906. https://doi.org/10.1094/PDIS-08-11-0689-PDN. 3. Crous, P. W., et al. 2006. Stud. Mycol. 55:235. https://doi.org/10.3114/sim.55.1.235 4. Ezra et al. 2013. Plant Disease 97: 1513. https://doi.org/10.1094/PDIS-05-13-0535-PDN 5. Lan, G.B. et al. 2012. Plant Disease 96: 1702. https://doi.org/10.1094/PDIS-07-12-0632-PDN 6. Sanahuja et al. 2016. Plant Disease 100: 1499. https://doi.org/10.1094/PDIS-11-15-1319-PDN 7. White, T., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

Diaphorina citri (Hemiptera: Liviidae) Abundance in Puerto Rico Declines with Elevation.

Diaphorina citri Kuwayama is the primary vector of Huanglongbing, the most devastating disease of citrus. D. citri populations in Puerto Rico were monitored with yellow sticky traps on citrus trees or other psyllid host plants at different elevations, ranging from 10 to 880 m above sea level. Trapping was conducted in March through May of 2013 and 2014 when psyllid populations usually are highest. Population levels of D. citri, based on the trapping data, varied among the sites, and there was a strong trend in both years for decreasing psyllid abundance with increased elevation based on the number of psyllids captured on traps and the proportion of trees shown to be infested. No psyllids were collected at an elevation of >600 m. Reduced populations at higher elevations could be a consequence of differences in temperature, air pressure, oxygen levels, ultraviolet light, or other factors alone or in combination. We discuss our results as they pertain to management of D. citri and Huanglongbing.

Attraction of Pollinators to Atemoya (Annona squamosa × Annona cherimola) in Puerto Rico Using Commercial Lures and Food Attractants.

Atemoya is a hybrid between Annona squamosa L. and Annona cherimola Miller (Annonaceae) and has potential to be an important fruit crop in tropical and subtropical areas. A major impediment to fruit production is low fruit set due to inadequate pollinator visits, typically, by beetles in the family Nitidulidae. We used Universal moth traps to monitor the attractiveness of two commercially available Nitidulidae lures in combination with various food attractants, including raw bread dough, apple juice, and malta beverage, a soft drink by-product of the brewing process. The most commonly trapped beetles were, in order of decreasing frequency, Carpophilus dimidiatus (F.), Brachypeplus mutilatus Erichson, Urophorus humeralis (F.) (Coleoptera: Nitidulidae), and Europs fervidus Blatchley (Coleoptera: Monotomidae). All traps, except the unbaited control traps, caught beetles. In a previous study, we found that combining two commercial lures had a synergistic effect on the attraction of these beetle species. In this study, the addition of food attractants increased the number of beetles trapped compared with traps baited with only the commercial lures. Also, food attractants appear to be key in attracting U. humeralis; only one U. humeralis individual of the 206 caught during the experiment was trapped without a food attractant. The variation between the number of beetles caught in traps containing the same treatments was high and may explain the erratic results reported in other studies of pollination in Annona spp. The results are discussed with respect to the use of nitidulid lures and food attractants to increase fruit set in atemoya and other Annonaceae.

The American Cherimoya Genome Reveals Insights into the Intra-Specific Divergence, the Evolution of Magnoliales, and a Putative Gene Cluster for Acetogenin Biosynthesis.

Annona cherimola (cherimoya) is a species renowned for its delectable fruit and medicinal properties. In this study, we developed a chromosome-level genome assembly for the cherimoya 'Booth' cultivar from the United States. The genome assembly has a size of 794 Mb with a N50 = 97.59 Mb. The seven longest scaffolds account for 87.6% of the total genome length, which corresponds to the seven pseudo-chromosomes. A total of 45,272 protein-coding genes (≥30 aa) were predicted with 92.9% gene content completeness. No recent whole genome duplications were identified by an intra-genome collinearity analysis. Phylogenetic analysis supports that eudicots and magnoliids are more closely related to each other than to monocots. Moreover, the Magnoliales was found to be more closely related to the Laurales than the Piperales. Genome comparison revealed that the 'Booth' cultivar has 200 Mb less repeats than the Spanish cultivar 'Fino de Jete', despite their highly similar (>99%) genome sequence identity and collinearity. These two cultivars were diverged during the early Pleistocene (1.93 Mya), which suggests a different origin and domestication of the cherimoya. Terpene/terpenoid metabolism functions were found to be enriched in Magnoliales, while TNL (Toll/Interleukin-1-NBS-LRR) disease resistance gene has been lost in Magnoliales during evolution. We have also identified a gene cluster that is potentially responsible for the biosynthesis of acetogenins, a class of natural products found exclusively in Annonaceae. The cherimoya genome provides an invaluable resource for supporting characterization, conservation, and utilization of Annona genetic resources.

Attraction of pollinators to atemoya (Magnoliales: Annonaceae) in Puerto Rico: a synergistic approach using multiple nitidulid lures.

Atemoya, a hybrid between Annona squamosa (L.) and A. cherimola Miller (Annonaceae), has potential to be a major fruit crop in tropical and subtropical areas. A major setback to fruit production throughout the world is low fruit-set because of inadequate pollinator visits, typically Nitidulidae beetles. We identified beetle visitors to atemoya flowers in an orchard in Puerto Rico and used Universal moth traps to monitor the attractiveness of two commercially available Nitidulidae lures. The most common visitors to atemoya flowers were an unidentified Europs species (Coleoptera: Monotomidae), followed by Loberus testaceus (Coleoptera: Erotylidae), neither of which have been previously reported as visitors to Annona flowers. The commercial lures attracted few or no beetles when used separately, but attracted a large number of beetles, especially Carpophilus dimidiatus (Coleoptera: Nitidulidae) and Europs, when used in combination. This attraction is synergistic and increases with dose at the doses assayed (0-4 lures), and decreases over time with >50% of trap captures occurring in the first week and no beetles collected after 5 wk. This is the first report of aggregation pheromone lures in nitidulids acting synergistically to attract other species, including beetles not in the Nitidulidae. The results are discussed as they pertain to increasing fruit set, as well as the potential for altering fruit size and shape in atemoya.

The chromosome-level genome of dragon fruit reveals whole-genome duplication and chromosomal co-localization of betacyanin biosynthetic genes.

Dragon fruits are tropical fruits economically important for agricultural industries. As members of the family of Cactaceae, they have evolved to adapt to the arid environment. Here we report the draft genome of Hylocereus undatus, commercially known as the white-fleshed dragon fruit. The chromosomal level genome assembly contains 11 longest scaffolds corresponding to the 11 chromosomes of H. undatus. Genome annotation of H. undatus found ~29,000 protein-coding genes, similar to Carnegiea gigantea (saguaro). Whole-genome duplication (WGD) analysis revealed a WGD event in the last common ancestor of Cactaceae followed by extensive genome rearrangements. The divergence time between H. undatus and C. gigantea was estimated to be 9.18 MYA. Functional enrichment analysis of orthologous gene clusters (OGCs) in six Cactaceae plants found significantly enriched OGCs in drought resistance. Fruit flavor-related functions were overrepresented in OGCs that are significantly expanded in H. undatus. The H. undatus draft genome also enabled the discovery of carbohydrate and plant cell wall-related functional enrichment in dragon fruits treated with trypsin for a longer storage time. Lastly, genes of the betacyanin (a red-violet pigment and antioxidant with a very high concentration in dragon fruits) biosynthetic pathway were found to be co-localized on a 12 Mb region of one chromosome. The consequence may be a higher efficiency of betacyanin biosynthesis, which will need experimental validation in the future. The H. undatus draft genome will be a great resource to study various cactus plants.

Host breadth and parasitoids of fruit flies (Anastrepha spp.) (Diptera: Tephritidae) in Puerto Rico.

Twenty fruit species representing 12 families were collected from various regions in western Puerto Rico and monitored for the emergence of Anastrepha spp. pupae. We collected 14,154 tephritid pupae from 16 fruit species representing 10 families. The relative infestations of these fruits (pupae per kilogram of fruit) were recorded. Recorded host ranges were not in complete agreement with those reported in the literature. This host-use pattern should give pause to regulators of fruit importation and exportation that base their decisions on literature from regions other than those of immediate interest to them. We recovered the braconid parasitoid Utetes anastrephae (Viereck) from tephritid pupae collected from Mangifera indica L., Spondias mombin L., Psidium guajava L., Chrysobalanus icacos L., Terminalia catappa L., and Garcinia intermedia (Pittier) Hammel. We collected one specimen of the parasitoid Doryctobracon aerolatus (Szepligeti) from the west coast (Añasco), which had not been previously reported in Puerto Rico. We present a preliminary phenology of what are probably the primary fruit hosts of the Anastrepha spp. of Puerto Rico. We also present the first report of Garcinia intermedia (Pittier) Hammel and Coffea arabica L. as reproductive hosts of A. suspensa.

Forest fragments as barriers to fruit fly dispersal: Anastrepha (Diptera: Tephritidae) populations in orchards and adjacent forest fragments in Puerto Rico.

McPhail-type traps baited with ammonium acetate and putrescine were used to monitor populations of Anastrepha obliqua (Macquart) and Anastrepha suspensa (Loew) in two orchards with hosts of these flies (mango, Mangifera indica L., and carambola, Averrhoa carambola L.), as well as in forest fragments bordering these orchards. Contour maps were constructed to measure population distributions in and around orchards. Our results indicate that Anastrepha populations are focused around host fruit in both space and time, that traps do not draw fruit flies away from hosts, even when placed within 15 m of the host, and that lures continue to function for 6 mo in the field. The contour mapping analyses reveal that populations of fruit flies are focused around ovipositional hosts. Although the trapping system does not have a very long effective sampling range, it is ideal, when used in combination with contour analyses, for assessing fine-scale (on the order of meters) population distributions, including identifying resources around which fly populations are focused or, conversely, assessing the effectiveness of management tools. The results are discussed as they pertain to monitoring and detecting Anastrepha spp. with the McPhail-type trap and ammonium acetate and putrescine baiting system and the dispersal of these flies within Puerto Rico.

Dolabra nepheliae on rambutan and lychee represents a novel lineage of phytopathogenic Eurotiomycetes.

Rambutan (Nephelium lappaceum) and lychee (Litchi chinensis) are tropical trees in the Sapindaceae that produce delicious edible fruits and are increasingly cultivated in tropical regions. These trees are afflicted with a stem canker disease associated with the ascomycete Dolabra nepheliae. Previously known from Asia and Australia, this fungus was recently reported from Hawaii and Puerto Rico. The sexual and asexual states of Dolabra nepheliae are redescribed and illustrated. In addition, the ITS and large subunit of the nuclear ribosomal DNA plus fragments from the genes RPB2, TEF1, and the mitochondrial small ribosomal subunit were sequenced for three isolates of D. nepheliae and compared with other sequences of ascomycetes. It was determined that D. nepheliae represents a new lineage within the Eurotiomycetes allied with Phaeomoniella chlamydospora, the causal agent of Petri grapevine decline.