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.

The Sigatoka Disease Complex Caused by Pseudocercospora spp. and Other Fungal Pathogens Associated with Musa spp. in Puerto Rico.

Bananas (Musa spp.) are among the world's most economically important staple food crops. The most important fungal leaf diseases of Musa spp. worldwide are caused by the Sigatoka disease complex, which comprise black Sigatoka (Pseudocercospora fijiensis), yellow Sigatoka (P. musae), and Eumusae leaf spot (P. eumusae). Considering the rapid spreading rate of black Sigatoka in Puerto Rico after its first observation in 2004, a disease survey was conducted from 2018 to 2020 to evaluate the Sigatoka disease complex on the island. Sixty-one leaf samples showing Sigatoka-like symptoms were collected throughout the island for diagnosis by molecular approaches and fungal isolation. Molecular analysis using species-specific primers for P. fijiensis, P. musae and P. eumusae detected the presence of P. fijiensis in fifty leaf samples. Thirty-eight fungal isolates were collected and identified by morphology and genomic sequencing from various nuclear genes. The analysis identified 24 isolates as P. fijiensis, while the rest of the isolates belonged to the genus Cladosporium spp. and Cladosporium-like spp. (n=5), Neocordana musae (n=2), Zasmidium spp. (n=6), and Z. musigenum (n=1). The high frequency of P. fijiensis found in leaf samples and collected isolates suggest that black Sigatoka has displaced the yellow Sigatoka (P. musae) in Puerto Rico. Accurate identification of fungal species causing foliar diseases in Musa spp. will allow the establishment of quarantine regulations and specific management approaches in Puerto Rico.

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.

First report of Lasiodiplodia mahajangana causing canker of mango (Mangifera indica) in Puerto Rico.

Mango originated in the Indo-Burmese region (Alphonse de Candolle, 1885). In the Caribbean, Puerto Rico currently produces and exports mangoes to the United States and Europe. Globally, an important disease affecting mango production is dieback, caused by fungi belonging to Botryosphaeriaceae family. During a one-year survey from 2019 to 2020, conducted at the mango germplasm collection of the Agricultural Experiment Station of the University of Puerto Rico, located at Juana Díaz, PR, symptoms of dieback were observed in shoots, descending towards the woody part, and vascular necrosis. We sampled bimonthly, 35 Keitt trees for one year. At the end of the evaluation, we detected that a 74% disease incidence was caused by Botryosphaeriaceae. Lasiodiplodia mahajangana (syn. L. caatinguensis) was associated with 4% disease incidence. In addition, we identified other Botryosphaeriaceae species causing 70% of disease incidence. To identify the causal agent, sections of symptomatic tissue (4mm2) were surface disinfected by immersion in 70% ethanol, 10% sodium hypochlorite and rinsed with sterile-distilled water for 1 minute at each solution. Sections were transferred to petri dishes containing potato dextrose agar acidified with 85% lactic acid (aPDA). Ten fungal isolates were obtained with similar morphological characteristics such as colony color and texture, after 12 days. Of these, one representative (isolate 17) was selected and identified as L. mahajangana (Lm) using morphological parameters and sequences of four nuclear genes (Zhang, W. et al., 2021). In aPDA, Lm colonies showed sparse and slow-growing aerial mycelium with dark gray-greenish color at the center and light gray edges. Black pycnidia were observed after 15 days of incubation at 28°C and dark conditions. Hyaline, ovoid to ellipsoid immature conidia (n=40) with average size of 22 µm long and 12 µm wide were observed. Mature bicellular pigmented conidia (n=40) had longitudinal striate and its average size was 23 µm long and 12 µm wide. Internal transcribed spacer (ITS), ß-tubulin (ßtub), elongation factor 1-alpha (EF1-α) and large ribosomal subunit (LSU) genetic regions were amplified by PCR from the original and pathogenicity test recovered isolates. Sequences of PCR products were compared with NCBI database BLAST tool with other Lm sequences. Sequence accession numbers of the four genetic regions of Lm are as follows: OL375401 and OL375402 for the ITS region; OL405579 and OL405580 for ß-tubulin; OL455922 and OL455923 for EF1-α; and OL375648 and OL375649 for LSU. All the sequences were grouped with the ex-type CMM1325 of Lm (BS=84). Pathogenicity tests were performed on 6-month-old mango trees of cv. Keitt. Three healthy trees were inoculated with 5 mm mycelial disks of Lm, on stems, with and without wounds. Controls were inoculated with aPDA disks only. Inoculated trees were covered for 3 days with plastic bags, keeping them in conditions of high relative humidity with constant irrigation, temperature of 28°C, and 12 hours of light and 12 hours of darkness for 12 days. Twelve days after inoculation, Lm isolates caused stem necrosis and canker, with differences in lesion severity from 2 to 17 mm2 with wound, and 0 to 6 mm2 without wound. Untreated controls showed no symptoms of canker. Lasiodiplodia mahajangana was re-isolated from diseased stems fulfilling Koch's postulates, and a sequence of the recovered isolate from the pathogenicity test was compared and included in the phylogenetic analysis. Lasiodiplodia mahajangana has been reported to cause stem-end rot of mango in Malaysia (Li, L. et. al., 2021). To our knowledge, this is the first report of Lm causing canker of mango in Puerto Rico. Knowing L. mahajangana as a new pathogen that causes canker of mango is important to establish an adequate and effective control management of this disease in mango producing countries worldwide.

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.

Pathogens Causing Anthracnose and Fruit Rots of Coffee Associated with the Coffee Berry Borer and the Entomopathogenic Fungus Beauveria bassiana in Puerto Rico.

Fruit rots reduce coffee production worldwide. Eight Colletotrichum species have been reported to cause coffee fruit rots; the most important is C. kahawae, the cause of coffee berry disease (CBD) in Africa. It is unknown whether these fruit rot pathogens can be dispersed by the coffee berry borer (CBB, Hypothenemus hampei) or whether Beauveria bassiana (a natural enemy of CBB) might reduce coffee fruit rots. We identified pathogens causing coffee fruits rots in Puerto Rico and evaluated whether B. bassiana reduced fruit rot and whether CBB could disperse pathogens. A total of 2,333 coffee fruit with CBB damage were collected; of these, 1,197 had visible growth of B. bassiana. C. fructicola, C. siamense, C. theobromicola, and C. tropicale were isolated and identified from the fruit using morphological traits and phylogeny of three nuclear genes. All four species caused internal and external rot after inoculation of healthy green coffee fruit. Coffee fruit treated with B. bassiana had significantly less fruit rot than untreated fruit, suggesting B. bassiana can protect against fruit rot. To test whether B. bassiana had a protective effect, B. bassiana and Colletotrichum were coinoculated on coffee fruit. Fruit inoculated with both B. bassiana and Colletotrichum had significantly less rot than fruit inoculated with Colletotrichum alone. To test if CBBs dispersed the pathogens, CBBs were exposed to Colletotrichum conidia and placed on green fruit, which resulted in fruit rot. This study identifies new pathogens causing coffee fruit rot, shows that C. kahawae is not the only Colletotrichum that attacks green fruits, suggests a role for B. bassiana in disease management and demonstrates CBB can disperse the pathogens.

Forensic Phytopathology: a Critical Review.

Forensic phytopathology is the application of plant pathology to legal or criminal matters. It is an emerging field. The existing literature focuses mainly on potential agricultural bioterrorism threats to the United States. Here we try to take a broader view including agricultural bioterrorism, mycoherbicide applications to eradicate plants used for illegal drugs, civil cases involving charges of sale or movement of diseased plants, and mycotoxins. In several of the examples given the evidence is inconclusive, but the examples are no less interesting for that.