Lasiodiplodia theobromae disease symptom development in young avocado (Persea americana L.) plants depends on the inoculation method

Lasiodiplodia theobromae disease symptom development in young avocado (Persea americana L.) plants depends on the inoculation method | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Lasiodiplodia theobromae disease symptom development in young avocado (Persea americana L.) plants depends on the inoculation method Edgar Rodríguez-Gálvez, Cesar Haro-Diaz, Samir Maza-Aguirre, Fátima Canahuire-Castillo, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4496463/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Lasiodiplodia theobromae is a pathogenic fungus of more than 500 plant species, including avocado ( Persea americana Mill.). The global production volume of avocado exceeded 911,000 metric tons in 2023 (Statista, 2024). Although detailed quantitative surveys of yield losses caused by L. theobromae are not available for most avocado-producing regions, estimates indicate that this pathogen is associated with stem-end rot disease in 30–35% of plots located in the Department of Antioquia in Colombia (Ramírez-Gil et al., 2021 ), suggesting an enormous economic impact. As yield losses strongly depend on the virulence of isolates, a reliable method for determining their aggressiveness is indispensable for initiating disease control measures. In this study, we compared progression of external and internal necrosis caused by the aggressive L. theobromae isolate LA-VLCA3 inoculated into wounded middle parts of the stem and onto excised apices. Irrespective of the inoculation method, internal progression of necroses preceeded that of external necroses. Spreading of external and internal necroses was significantly more severe in plants inoculated at the apex than in mid-stem inoculated plants. We conclude that apex inoculation causes stronger symptoms and is therefore better suited to compare virulence of isolates than mid-stem inoculation. Avocado external and internal necroses Lasiodiplodia theobromae Persea americana symptom development virulence Figures Figure 1 Figure 2 Figure 3 Full Text The polyphagous pathogen Lasiodiplodia theobromae (Pat.) Griffon and Maubl. (1909) belongs to the family Botryosphaeriaceae (Dissanayake et al., 2016; Zhang et al., 2021) and causes multiple disease symptoms on approx. 500 plants (Punithalingam, 1976). In Peru, this pathogen has been described as a causal agent of dieback and wood necrosis in blueberry (Rodríguez-Gálvez et al., 2020), mango (Rodríguez-Gálvez et al., 2017), avocado (Rodríguez-Gálvez et al., 2021) and table grapes (Rodríguez-Gálvez et al., 2015). As symptom severity and yield losses strongly depend on the virulence of isolates (Gunamalai et al. 2023), a reliable method for determining their aggressiveness is indispensable for performing efficient disease control measures. Virulence tests with L. theobromae isolates have been carried out using two methods, i.e. inoculation into the middle part of the stem after wounding (Bautista-Cruz et al., 2019; Berraf-Tebbal et al., 2020; Dong et al., 2020; Hernández et al., 2023; Úrbez-Torres et al., 2008; Úrbez-Torres et al., 2009; El-Ganainy et al., 2022; Coutinho et al., 2017; Rangel-Montoya et al., 2021; Briste et al., 2022; Kong et al., 2023), or onto excised apical bud of the stem (Alama et al., 2006; Billones-Baaijens et al., 2013; Hernández et al., 2023; Kwon et al., 2017; Polashock and Kramer, 2006; Rodríguez-Gálvez et al., 2015; Rodríguez-Gálvez et al., 2017; Rodríguez-Gálvez et al., 2020 and Rodríguez-Gálvez et al., 2021). In spite of the fact that distinct inoculation methods have been employed, rigorous comparisons of these methods have not been carried out. The present study was performed to determine which of the two inoculation methods yields more severe dieback and wood necroses symptoms and is therefore more suitable to determine virulence of isolates. In this study, 10 months old avocado plants ( Persea americana cv. Hass) plants grafted on West Indian rootstock were inoculated with the virulent L. theobromae isolate LA-VLCA3 obtained from avocado branches with dieback symptoms (Rodríguez-Gálvez et al., 2021). This isolate was plated onto 2% (w/v) potato dextrose agar (PDA) (HiMedia Laboratories Pvt.Ltd., Dindhori, Nashik, India) and incubated at 30°C for 72 h. Plants were inoculated using L. theobromae -covered agar blocks taken from the edge of a colony. This inoculation method allowed comparison of the data with those obtained in previous experiments (e.g. Rodríguez-Gálvez, et al 2015, 2017, 2020, 2021; Úrbez-Torres and Gubler, 2009; Úrbez-Torres at al. 2008). Moreover, we decided to use agar block inoculation because inoculation of conidial suspensions into wounds in vertical stems may result in major loss of conidia due to the outflow of an unknown volume of the inoculum. Mid-stem inoculation was done after wounding with a sterile corkborer (5 mm diameter) immediately above the grafting zone. An agar disc with mycelium (4 mm diameter) of the pathogen obtained from the edge of a colony at 72 hours post inoculation (hpi) was deposited into the wound and the inoculated area was covered with Parafilm (Bemis Company, Inc., Neenah, Wisconsin, USA) (Úrbez-Torres et al., 2008). For apex inoculation, the apical bud was cut off with a sterile scalpel and a 4 mm agar disc with fungal mycelium (see above) was placed onto the wound. The inoculated area was covered with sterile cotton moistened with sterile distilled water and sealed with Parafilm (Bemis Company, Inc., Neenah, Wisconsin, USA) (Rodríguez-Gálvez et al., 2015). Five plants per treatment were inoculated and incubated for 28 days in a greenhouse at an average temperature of 26°C. The experiment was repeated four times, yielding a total of 20 tested plants per treatment. Symptom development was observed daily and the expansion of external and internal necroses caused by the pathogen was assessed at 28 days post inoculation (dpi) by measuring length of necroses from the point of inoculation to the border of the visible infection using a digital vernier. In mid stem-inoculated plants, acropetal and basipetal external and internal necroses were measured, and in plants inoculated at the excised apex, only external and internal basipetal necroses were measured. To confirm statistical normality of necrosis progression, the SPSS-V.25 software (IBM, New York, USA) was used, applying the Shapiro-Wilk test for data smaller than 50. For comparison of means in samples that do not show normal data distribution, the non-parametric Kruskal-Wallis test was used. For comparison of means in samples from normal populations, the parametric ANOVA test was used, followed by Tukey's test (p ≤ 0.05). Statgraphics Centurion software version VII (Statgraphics Technologies Inc., The Plains, VA, USA) was employed for both comparisons. In plants inoculated at the mid-stem, an irregular externally visible black necrotic spot was observed around the inoculation site. Developing necrosis expanded acro- and basipetally from the inoculation site (Figure 1a, arrowhead). After dissecting the cortical zone longitudinally, internal necrosis of the tissue was observed, which had a greater acropetal and basipetal extension than the externally visible necrosis (Figure 1b, arrows). Interestingly, cross-sections and longitudinal sections revealed that internal necroses first developed acro- and basipetally underneath the bark (Figure 1c, arrows), established bark-associated necroses and subsequently colonized the xylem and grew towards the pith, resulting in the sectorial necrosis typically observed in L. theobromae -infected stems (Figure 1d, arrows). Thus, development of necroses in avocado resembles those observed in L. theobromae -infected grapes (Rodríguez-Gálvez, 2021). Following apex inoculation, necroses developed dramatically and expanded basipetally, affecting all stem tissues from the point of inoculation, and resulted in necrotizing leafs and branches at 28 dpi (Figure 2a). Formation of enormous numbers of conidia covering a large part of the infected stem was visible as whitish coating (Figures 2a and, at larger magnification, Figure 2b, co). Internal necrosis extending basipetally beyond the edge of the externally visible necrosis (Figure 2c, arrowhead) was detected after cutting the bark longitudinally (Figure 2c, arrows). Macroscopic evaluation of infected plants (Figures 1 and 2), and quantification of the length of external and internal necroses revealed that the fungus had massively spread in the stem (internal necroses) before symptoms become externally visible (Figure 3). In mid-stem-inoculated plants, no significant differences were observed between acropetal and basipetal expansion of necroses (p = 0.432774 > ɑ=0.05), indicating similar fungal upward- and downward-directed spread. Importantly, basipetal extension of both external and internal necroses were significantly more pronounced in stems inoculated at the excised apex, as compared with mid stem-inoculated plants (p=000000006318 < ɑ=0.05) (Figure 3). Clearly, these data indicate that excised-stem-inoculation is an excellent method to evaluate virulence of field isolates of L. theobromae . Our results show that development of necroses in stems of young avocado plants inoculated with L. theobromae is strongly affected by the inoculation method used. The advance of internal necrotization preceeded that of external necrotization, suggesting that the fungus spreads vertically within the stem before expressing external disease symptoms. Interestingly, basipetal spread of the fungus as well as generation of internal and external necroses occurred more efficiently after inoculation of the excised apex than after mid-stem inoculation. In comparison, Úrbez-Torres et al. (2008) reported that L. theobromae caused larger basipetal than acropetal lesions in rooted cuttings of grapevine cv. Chardonnay and cv. Thompson, and on green shoots of grapevine cvs. Chardonnay and Thompson. Reports of these two types of colonization were also addressed in other research studies on wood colonization by various plant pathogenic fungi, including L. theobromae (Úrbez-Torres et al, 2009 and Bautista-Cruz et al., 2019); L. crassispora , L. euphorbicola and L. pseudotheobromae (Dianda et al., 2023), Neofusicoccum luteum and N. parvum (Billones-Baaijens et al., 2013). The differences between external and internal colonization addressed in this study have not been considered in other investigations yet. In most of the previous studies only internal colonization has been studied (Berraf-Tebbal et al., 2020; Biju, et al., 2021; Briste et al., 2022; Dianda et al., 2023; Dong et al., 2020; El-Ganainy et al., 2022; Gunamalai, et al., 2023; Hernandez et al., 2023). In avocado, we measured a sum of acropetal plus basipetal internal necrosis of 230 mm in only 28 days of incubation. In comparison, Úrbez-Torres et al. (2008) calculated a lesion length of 338 mm when inoculating rooted cuttings of grapevine cv. Chardonnay and 183.1 mm in cv. Red Globe, with an incubation time as long as of 140 days. The same authors obtained lower values in green shoots of grapevine cv. Red Globe and in rooted cuttings of this cultivar after an incubation time of 180 days (Úrbez-Torres et al., 2009). Li et al. (2013), report lesion lengths of 149.1 mm in grapevine cv. Chardonnay incubated for 720 days. Rapid generation of significant external and internal necroses caused by L. theobromae observed after inoculation of the excised apex indicates that this method is not only suitable for quantitative determination of virulence of field isolates of this fungus, but could also be an excellent and time-saving alternative in testing virulence of isolates of other members of the Botryosphaeriaceae . Declarations Authors' contribution statement : The conceptualisation of this research was developed by E. Rodríguez-Gálvez (Principal Investigator) and Holger Deising. The experimental design and statistical analyses were developed by C. Haro-Díaz, the maintenance of the plants and the preparation of the virulent isolate LA-VLCA3 of L. theobromae for inoculation was performed by S. Maza-Aguirre. Inoculation of plants and evaluation of symptoms was performed by J. Sullón-Saucedo and F. Canahuire-Castillo. E. Rodríguez-Gálvez analyzed the data, and E. Rodríguez-Gálvez and Holger B. Deising wrote the manuscript. Revision of the article was performed by all authors. Data availability : All data generated and analyzed are available upon request to the corresponding author. Conflicts of Interest : The authors declare no conflict of interest. Acknowledgements : The authors would like to thank Technician Angela Peña Gonzales for her excellent technical assistance and the “Los Viñedos” nursery for the donation of the avocado seedlings. This work was financed by the Central Research Office (OCIN) of the National University of Piura through the University Research Fund (FEDU). References Alama I, Maldonado E, Rodríguez-Gálvez E (2006) Lasiodiplodia theobromae afectando el Cultivo de Palto ( Persea americana ) en las condiciones de Piura-Perú. Universalia 11:4–13 Bautista-Cruz MA, Almaguer-Vargas G, Leyva-Mir SG, Colinas-León MT, Correia KC, CamachoTapia M, Robles-Yerena L, Tovar-Pedraza JM (2019) Phylogeny, distribution, and pathogenicity of Lasiodiplodia species associated with cankers and dieback symptoms of Persian lime in Mexico. Plant Dis 103:1156–1165 Berraf-Tebbal A, Mahamedi AE, Aigoun-Mouhous W, Špetík M, Čechová J, Pokluda R, Baránek M, Eichmeier A, Alves A (2020) Lasiodiplodia mitidjana sp. nov. and other Botryosphaeriaceae species causing branch canker and dieback of Citrus sinensis in Algeria. PLoS ONE 15:e0232448 Biju CN, Jeevalatha A, Peeran MF, Bhai RS, Basima F, Nissar VM, Srinivasan V, Thomas L (2021) Association of Lasiodiplodia theobromae with die-back and decline of nutmeg as revealed through phenotypic, pathogenicity and phylogenetic analyses. 3 Biotech 11:422 Billones-Baaijens R, Jones EE, Ridgway HJ, Jaspers MV (2013) Virulence affected by assay parameters during grapevine pathogenicity studies with Botryosphaeriaceae nursery isolates. Plant Pathol 62:1214–1225 Briste PS, Akanda AM, Bhuiyan MAB, Mahmud NU, Islam T (2022) Morphomolecular and cultural characteristics and host range of Lasiodiplodia theobromae causing stem canker disease in dragón fruit. J Basic Microbiol 62:689–700 Coutinho IBL, Freire FCO, Lima CS, Lima JS, Gonçalves FJT, Machado AR, Silva AMS, Cardoso JE (2017) Diversity of genus Lasiodiplodia associated with perennial tropical fruit plants in northeastern Brazil. Plant Pathol 66:90–104 Dianda OZ, Issa W, Ouédraogo L, Sankara P, Tollenaere C, Del Ponte EM, Fernandez D (2023) Lasiodiplodia species associated with mango ( Mangifera indica L.) decline in Burkina Faso and influence of climatic factors on the disease distribution. Physiol Mol Plant Pathol 126:1020412023–1020412003 Dissanayake AJ, Phillips AJL, Li XH, Hyde KD (2016) Botryosphaeriaceae : Current status of genera and species. Mycosphere 7:1001–1073 Dong W, Doilom M, Hyde KD, Phillips A, Yan K, To-anun C, Xu JC, Zhang H, Nalumpang S (2020) Pathogenicity of five Botryosphaeriaceae species isolated from Tectona grandis (teak): the pathogenic potential of Lasiodiplodia species. Asian J Mycol 3:399–407 El-Ganainy SM, Ismail AM, Iqbal Z, Elshewy ES, Alhudaib KA, Almaghasla MI, Magistà D (2022) Diversity among Lasiodiplodia Species Causing Dieback, Root Rot and Leaf Spot on Fruit Trees in Egypt, and a Description of Lasiodiplodia newvalleyensis sp. nov. J Fungi 8:1203 Gunamalai L, Duanis-Assaf D, Maurer D, Sharir T, Feigenberg O, Sela N, Alkan N (2023) Comparative characterization of virulent and less virulent Lasiodiplodia theobromae isolates. Mol Plant Microbe Interact 36:502–515 Hernández D, García-Pérez O, Perera S, González-Carracedo MA, Rodríguez-Pérez A, Siverio F (2023) Fungal pathogens associated with aerial symptoms of avocado ( Persea americana Mill.) in Tenerife (Canary Islands, Spain) focused on species of the family Botryosphaeriaceae . Microorganisms 11:585 Kwon JH, Choi O, Kang B, Lee Y, Park J, Kang DW, Han I, Kim J (2017) Identification of Lasiodiplodia pseudotheobromae causing mango dieback in Korea. Can J Plant Pathol 39:241–245 Kong Q, Xing S, Xie K, Jia Z, Li Z, Liu Y, Xu C, Shi X, Wang C, Yuan S (2023) First report of Lasiodiplodia theobromae causing stem brown rot of loquat in China. Plant Dis 107:2234 Polashock JJ, Kramer M (2006) Resistance of blueberry cultivars to Botryosphaeria stem blight and Phomopsis twig blight. Hort Sci 41:1457–1461 Punithalingam E (1976) Botryodiplodia theobromae . CMI Descriptions of Fungi and Bacteria: 52, Sheet-519. Commonwealt Mycological Institute, Kew, Surrey, England Rangel-Montoya EA, Paolinelli M, Rolshausen PE, Valenzuela-Solano C, Hernandez-Martinez R (2021) Characterization of Lasiodiplodia species associated with grapevines in Mexico. Phytopathologia Mediterranea 60:237–251 Ramírez-Gil JG, Henao-Rojas JC, Morales-Osorio JG (2021) Postharvest diseases and disorders in avocado cv. Hass and their relationship to preharvest management practices. Heliyon 7:e05905 Rodríguez-Gálvez E (2021) Enfermedades de la madera en los cultivos de Mango, Palto, Vid y Arándano, en Piura- Perú. Sintomatología y Etiología. Editorial Letra. 200pp Rodríguez-Gálvez E, Guerrero P, Barradas C, Crous PW, Alves A (2017) Phylogeny and pathogenicity of Lasiodiplodia species associated with dieback of mango in Peru. Fungal Biology 121:452–465 Rodríguez-Gálvez E, Hilário S, Batista E, Lopes A, Alves A (2021) Lasiodiplodia species associated with dieback of avocado in the coastal area of Peru. Eur J Plant Pathol 161:219–232 Rodríguez-Gálvez E, Hilário S, Lopes A, Alves A (2020) Diversity and pathogenicity of Lasiodiplodia and Neopestalotiopsis species associated with stem blight and dieback of blueberry plants in Peru. Eur J Plant Pathol 157:89–102 Rodríguez-Gálvez E, Maldonado E, Alves A (2015) Identification and pathogenicity of Lasiodiplodia theobromae causing dieback of table grapes in Peru. Eur J Plant Pathol 141:477–489 Statista Research Department https://es.statista.com/acercadenosotros/nuestro-compromiso-con-la-calidad Úrbez-Torres JR, Gubler WD (2009) Pathogenicity of Botryosphaeriaceae species isolated from grapevine cankers in California. Plant Dis 93:584–592 Úrbez-Torres JR, Leavitt GM, Guerrero JC, Guevara J, Gubler WD (2008) Identification and pathogenicity of Lasiodiplodia theobromae and Diplodia seriata , the causal agents of bot canker disease of grapevines in Mexico. Plant Dis 92:519–529 Zhang W, Groenewald JZ, Lombard L, Schumacher RK, Phillips AJL, Crous PW (2021) Evaluating species in Botryosphaeriales . Persoonia – Mol Phylogeny Evol Fungi 46:63–115 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 20 Jul, 2024 Reviewers invited by journal 17 Jul, 2024 Editor assigned by journal 09 Jul, 2024 First submitted to journal 02 Jul, 2024 Editorial decision: Major revisions 27 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4496463","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":328365094,"identity":"910ccc09-e2d6-4b0a-a37c-a0e336fbfe66","order_by":0,"name":"Edgar Rodríguez-Gálvez","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Edgar","middleName":"","lastName":"Rodríguez-Gálvez","suffix":""},{"id":328365095,"identity":"b2dece95-3f61-4397-8e0b-eb992333c37b","order_by":1,"name":"Cesar Haro-Diaz","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Cesar","middleName":"","lastName":"Haro-Diaz","suffix":""},{"id":328365096,"identity":"7fa4d20a-fe26-4496-980a-2d14784e8487","order_by":2,"name":"Samir Maza-Aguirre","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Samir","middleName":"","lastName":"Maza-Aguirre","suffix":""},{"id":328365097,"identity":"c2f7fe9e-7e1e-4068-9571-49800be8b681","order_by":3,"name":"Fátima Canahuire-Castillo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Fátima","middleName":"","lastName":"Canahuire-Castillo","suffix":""},{"id":328365098,"identity":"baa0e430-df85-4c67-b688-f26125c0c54f","order_by":4,"name":"Julio Sullón-Saucedo","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Julio","middleName":"","lastName":"Sullón-Saucedo","suffix":""},{"id":328365099,"identity":"44523eed-14f0-4a5f-8fff-fc8728138c05","order_by":5,"name":"Holger B. 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Hass inoculated with \u003cem\u003eL. theobromae\u003c/em\u003e in the mid-part of the stem. a, external necrosis. b, internal necrosis of the same inoculation site observable after longitudinal sectioning of the bark. Arrowheads in a and b indicate the inoculation point. c, longitudinal extension of surface-associated necroses (arrows). d, cross section showing horizontal spread of necrosis (arrows) from the bark towards the pith.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4496463/v1/be7eb7ad8d7a593a7d6f32ac.png"},{"id":62265401,"identity":"0925713c-bf3b-4c4a-80ed-54d64b172339","added_by":"auto","created_at":"2024-08-12 09:16:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1218445,"visible":true,"origin":"","legend":"\u003cp\u003eNecrosis symptom on avocado (\u003cem\u003ePersea americana\u003c/em\u003e) cv. Hass plant after apex inoculation with \u003cem\u003eL. theobromae\u003c/em\u003e. a, external apical necrosis, with an arrowhead marking the edge of external necrosis and bracket marking the area of conidiation. b, Enlargement of a, showing a whitish conidiation area (co) of the pathogen and arrowhead pointing at the edge of necrosis. c, Internal necrosis of the same infection site, observed after longitudinal sectioning of the bark. The arrowhead indicates the edge of the external necrotic area; note the downward extension of internal necrosis as indicated by arrows below arrowhead.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4496463/v1/00464069efdfa10251920b8e.png"},{"id":62265400,"identity":"9c336ef6-b0de-4794-85f7-91a10d4a1ed5","added_by":"auto","created_at":"2024-08-12 09:16:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":51975,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the length of external and internal necroses in plants wound-inoculated at the mid-stem or at the excised apex. In mid-stem-inoculated plants, acropetal and basipetal extension of external and internal necroses is shown. In apex-inoculated plants, only basipetal extension of external and internal necroses were measured. * indicates statistical significance (p ≤ 0.05).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4496463/v1/28aaede652a96021ec6f3e56.png"},{"id":62265403,"identity":"9eb1d489-815e-4475-88b5-bec7c32a0db9","added_by":"auto","created_at":"2024-08-12 09:16:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2853154,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4496463/v1/1cea6692-d400-4648-ab1f-2bc07a26fa53.pdf"}],"financialInterests":"","formattedTitle":"Lasiodiplodia theobromae disease symptom development in young avocado (Persea americana L.) plants depends on the inoculation method","fulltext":[{"header":"Full Text","content":"\u003cp\u003eThe polyphagous pathogen \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e (Pat.) Griffon and Maubl. (1909) belongs to the family \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e (Dissanayake et al., 2016; Zhang et al., 2021) and causes multiple disease symptoms on approx. 500 plants (Punithalingam, 1976). In Peru, this pathogen has been described as a causal agent of dieback and wood necrosis in blueberry (Rodr\u0026iacute;guez-G\u0026aacute;lvez et al., 2020), mango (Rodr\u0026iacute;guez-G\u0026aacute;lvez et al., 2017), avocado (Rodr\u0026iacute;guez-G\u0026aacute;lvez et al., 2021) and table grapes (Rodr\u0026iacute;guez-G\u0026aacute;lvez et al., 2015). As symptom severity and yield losses strongly depend on the virulence of isolates (Gunamalai et al. 2023), a reliable method for determining their aggressiveness is indispensable for performing efficient disease control measures. Virulence tests with \u003cem\u003eL. theobromae\u003c/em\u003e isolates have been carried out using two methods, i.e. inoculation into the middle part of the stem after wounding (Bautista-Cruz et al., 2019; Berraf-Tebbal et al., 2020; Dong et al., 2020; Hern\u0026aacute;ndez et al., 2023; \u0026Uacute;rbez-Torres et al., 2008; \u0026Uacute;rbez-Torres et al., 2009; El-Ganainy et al., 2022; Coutinho et al., 2017; Rangel-Montoya et al., 2021; Briste et al., 2022; Kong et al., 2023), or onto excised apical bud of the stem (Alama et al., 2006; Billones-Baaijens et al., 2013; Hern\u0026aacute;ndez et al., 2023; Kwon et al., 2017; Polashock and Kramer, 2006; Rodr\u0026iacute;guez-G\u0026aacute;lvez et al., 2015; Rodr\u0026iacute;guez-G\u0026aacute;lvez et al., 2017; Rodr\u0026iacute;guez-G\u0026aacute;lvez et al., 2020 and Rodr\u0026iacute;guez-G\u0026aacute;lvez et al., 2021). In spite of the fact that distinct inoculation methods have been employed, rigorous comparisons of these methods have not been carried out. The present study was performed to determine which of the two inoculation methods yields more severe dieback and wood necroses symptoms and is therefore more suitable to determine virulence of isolates.\u003c/p\u003e\n\u003cp\u003eIn this study, 10 months old avocado plants (\u003cem\u003ePersea americana\u003c/em\u003e cv. Hass) plants grafted on West Indian rootstock were inoculated with the virulent \u003cem\u003eL. theobromae\u003c/em\u003e isolate LA-VLCA3 obtained from avocado branches with dieback symptoms (Rodr\u0026iacute;guez-G\u0026aacute;lvez et al., 2021). This isolate was plated onto 2% (w/v) potato dextrose agar (PDA) (HiMedia Laboratories Pvt.Ltd., Dindhori, Nashik, India) and incubated at 30\u0026deg;C for 72 h.\u003c/p\u003e\n\u003cp\u003ePlants were inoculated using \u003cem\u003eL. theobromae\u003c/em\u003e-covered agar blocks taken from the edge of a colony. This inoculation method allowed comparison of the data with those obtained in previous experiments (e.g. Rodr\u0026iacute;guez-G\u0026aacute;lvez, et al 2015, 2017, 2020, 2021; \u0026Uacute;rbez-Torres and Gubler, 2009; \u0026Uacute;rbez-Torres at al. 2008). Moreover, we decided to use agar block inoculation because inoculation of conidial suspensions into wounds in vertical stems may result in major loss of conidia due to the outflow of an unknown volume of the inoculum. Mid-stem inoculation was done after wounding with a sterile corkborer (5 mm diameter) immediately above the grafting zone. An agar disc with mycelium (4 mm diameter) of the pathogen obtained from the edge of a colony at 72 hours post inoculation (hpi) was deposited into the wound and the inoculated area was covered with Parafilm (Bemis Company, Inc., Neenah, Wisconsin, USA) (\u0026Uacute;rbez-Torres et al., 2008). For apex inoculation, the apical bud was cut off with a sterile scalpel and a 4 mm agar disc with fungal mycelium (see above) was placed onto the wound. The inoculated area was covered with sterile cotton moistened with sterile distilled water and sealed with Parafilm (Bemis Company, Inc., Neenah, Wisconsin, USA) (Rodr\u0026iacute;guez-G\u0026aacute;lvez et al., 2015). Five plants per treatment were inoculated and incubated for 28 days in a greenhouse at an average temperature of 26\u0026deg;C. The experiment was repeated four times, yielding a total of 20 tested plants per treatment.\u003c/p\u003e\n\u003cp\u003eSymptom development was observed daily and the expansion of external and internal necroses caused by the pathogen was assessed at 28 days post inoculation (dpi) by measuring length of necroses from the point of inoculation to the border of the visible infection using a digital vernier. In mid stem-inoculated plants, acropetal and basipetal external and internal necroses were measured, and in plants inoculated at the excised apex, only external and internal basipetal necroses were measured.\u003c/p\u003e\n\u003cp\u003eTo confirm statistical normality of necrosis progression, the SPSS-V.25 software (IBM, New York, USA) was used, applying the Shapiro-Wilk test for data smaller than 50. For comparison of means in samples that do not show normal data distribution, the non-parametric Kruskal-Wallis test was used. For comparison of means in samples from normal populations, the parametric ANOVA test was used, followed by Tukey's test (p \u0026le; 0.05). Statgraphics Centurion software version VII (Statgraphics Technologies Inc., The Plains, VA, USA) was employed for both comparisons.\u003c/p\u003e\n\u003cp\u003eIn plants inoculated at the mid-stem, an irregular externally visible black necrotic spot was observed around the inoculation site. Developing necrosis expanded acro- and basipetally from the inoculation site (Figure 1a, arrowhead). After dissecting the cortical zone longitudinally, internal necrosis of the tissue was observed, which had a greater acropetal and basipetal extension than the externally visible necrosis (Figure 1b, arrows). Interestingly, cross-sections and longitudinal sections revealed that internal necroses first developed acro- and basipetally underneath the bark (Figure 1c, arrows), established bark-associated necroses and subsequently colonized the xylem and grew towards the pith, resulting in the sectorial necrosis typically observed in \u003cem\u003eL. theobromae\u003c/em\u003e-infected stems (Figure 1d, arrows). Thus, development of necroses in avocado resembles those observed in \u003cem\u003eL. theobromae\u003c/em\u003e-infected grapes (Rodr\u0026iacute;guez-G\u0026aacute;lvez, 2021).\u003c/p\u003e\n\u003cp\u003eFollowing apex inoculation, necroses developed dramatically and expanded basipetally, affecting all stem tissues from the point of inoculation, and resulted in necrotizing leafs and branches at 28 dpi (Figure 2a). Formation of enormous numbers of conidia covering a large part of the infected stem was visible as whitish coating (Figures 2a and, at larger magnification, Figure 2b, co). Internal necrosis extending basipetally beyond the edge of the externally visible necrosis (Figure 2c, arrowhead) was detected after cutting the bark longitudinally (Figure 2c, arrows).\u003c/p\u003e\n\u003cp\u003eMacroscopic evaluation of infected plants (Figures 1 and 2), and quantification of the length of external and internal necroses revealed that the fungus had massively spread in the stem (internal necroses) before symptoms become externally visible (Figure 3). In mid-stem-inoculated plants, no significant differences were observed between acropetal and basipetal expansion of necroses (p = 0.432774 \u0026gt; ɑ=0.05), indicating similar fungal upward- and downward-directed spread. Importantly, basipetal extension of both external and internal necroses were significantly more pronounced in stems inoculated at the excised apex, as compared with mid stem-inoculated plants (p=000000006318 \u0026lt; ɑ=0.05) (Figure 3). Clearly, these data indicate that excised-stem-inoculation is an excellent method to evaluate virulence of field isolates of \u003cem\u003eL. \u003c/em\u003e\u003cem\u003etheobromae\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eOur results show that development of necroses in stems of young avocado plants inoculated with \u003cem\u003eL. theobromae\u003c/em\u003e is strongly affected by the inoculation method used. The advance of internal necrotization preceeded that of external necrotization, suggesting that the fungus spreads vertically within the stem before expressing external disease symptoms. Interestingly, basipetal spread of the fungus as well as generation of internal and external necroses occurred more efficiently after inoculation of the excised apex than after mid-stem inoculation. In comparison, \u0026Uacute;rbez-Torres et al. (2008) reported that \u003cem\u003eL. theobromae\u003c/em\u003e caused larger basipetal than acropetal lesions in rooted cuttings of grapevine cv. Chardonnay and cv. Thompson, and on green shoots of grapevine cvs. Chardonnay and Thompson. Reports of these two types of colonization were also addressed in other research studies on wood colonization by various plant pathogenic fungi, including \u003cem\u003eL. theobromae\u003c/em\u003e (\u0026Uacute;rbez-Torres et al, 2009 and Bautista-Cruz et al., 2019); \u003cem\u003eL. crassispora\u003c/em\u003e, \u003cem\u003eL. euphorbicola\u003c/em\u003e and \u003cem\u003eL. pseudotheobromae\u003c/em\u003e (Dianda et al., 2023), \u003cem\u003eNeofusicoccum luteum\u003c/em\u003e and \u003cem\u003eN. parvum\u003c/em\u003e (Billones-Baaijens et al., 2013).\u003c/p\u003e\n\u003cp\u003eThe differences between external and internal colonization addressed in this study have not been considered in other investigations yet. In most of the previous studies only internal colonization has been studied (Berraf-Tebbal et al., 2020; Biju, et al., 2021; Briste et al., 2022; Dianda et al., 2023; Dong et al., 2020; El-Ganainy et al., 2022; Gunamalai, et al., 2023; Hernandez et al., 2023). In avocado, we measured a sum of acropetal plus basipetal internal necrosis of 230 mm in only 28 days of incubation. In comparison, \u0026Uacute;rbez-Torres et al. (2008) calculated a lesion length of 338 mm when inoculating rooted cuttings of grapevine cv. Chardonnay and 183.1 mm in cv. Red Globe, with an incubation time as long as of 140 days. The same authors obtained lower values in green shoots of grapevine cv. Red Globe and in rooted cuttings of this cultivar after an incubation time of 180 days (\u0026Uacute;rbez-Torres et al., 2009). Li et al. (2013), report lesion lengths of 149.1 mm in grapevine cv. Chardonnay incubated for 720 days.\u003c/p\u003e\n\u003cp\u003eRapid generation of significant external and internal necroses caused by \u003cem\u003eL. theobromae\u003c/em\u003e observed after inoculation of the excised apex indicates that this method is not only suitable for quantitative determination of virulence of field isolates of this fungus, but could also be an excellent and time-saving alternative in testing virulence of isolates of other members of the \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contribution statement\u003c/strong\u003e: The conceptualisation of this research was developed by E. Rodr\u0026iacute;guez-G\u0026aacute;lvez (Principal Investigator) and Holger Deising. The experimental design and statistical analyses were developed by C. Haro-D\u0026iacute;az, the maintenance of the plants and the preparation of the virulent isolate LA-VLCA3 of \u003cem\u003eL. theobromae\u003c/em\u003e for inoculation was performed by S. Maza-Aguirre. Inoculation of plants and evaluation of symptoms was performed by J. Sull\u0026oacute;n-Saucedo and F. Canahuire-Castillo. E. Rodr\u0026iacute;guez-G\u0026aacute;lvez analyzed the data, and E. Rodr\u0026iacute;guez-G\u0026aacute;lvez and Holger B. Deising wrote the manuscript. Revision of the article was performed by all authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: All data generated and analyzed are available upon request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e: The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: The authors would like to thank Technician Angela Pe\u0026ntilde;a Gonzales for her excellent technical assistance and the \u0026ldquo;Los Vi\u0026ntilde;edos\u0026rdquo; nursery for the donation of the avocado seedlings. This work was financed by the Central Research Office (OCIN) of the National University of Piura through the University Research Fund (FEDU).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlama I, Maldonado E, Rodr\u0026iacute;guez-G\u0026aacute;lvez E (2006) \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e afectando el Cultivo de Palto (\u003cem\u003ePersea americana\u003c/em\u003e) en las condiciones de Piura-Per\u0026uacute;. Universalia 11:4\u0026ndash;13\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBautista-Cruz MA, Almaguer-Vargas G, Leyva-Mir SG, Colinas-Le\u0026oacute;n MT, Correia KC, CamachoTapia M, Robles-Yerena L, Tovar-Pedraza JM (2019) Phylogeny, distribution, and pathogenicity of \u003cem\u003eLasiodiplodia\u003c/em\u003e species associated with cankers and dieback symptoms of Persian lime in Mexico. Plant Dis 103:1156\u0026ndash;1165\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBerraf-Tebbal A, Mahamedi AE, Aigoun-Mouhous W, Špet\u0026iacute;k M, Čechov\u0026aacute; J, Pokluda R, Bar\u0026aacute;nek M, Eichmeier A, Alves A (2020) \u003cem\u003eLasiodiplodia mitidjana\u003c/em\u003e sp. nov. and other \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e species causing branch canker and dieback of \u003cem\u003eCitrus sinensis\u003c/em\u003e in Algeria. PLoS ONE 15:e0232448\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBiju CN, Jeevalatha A, Peeran MF, Bhai RS, Basima F, Nissar VM, Srinivasan V, Thomas L (2021) Association of \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e with die-back and decline of nutmeg as revealed through phenotypic, pathogenicity and phylogenetic analyses. 3 Biotech 11:422\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBillones-Baaijens R, Jones EE, Ridgway HJ, Jaspers MV (2013) Virulence affected by assay parameters during grapevine pathogenicity studies with \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e nursery isolates. Plant Pathol 62:1214\u0026ndash;1225\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBriste PS, Akanda AM, Bhuiyan MAB, Mahmud NU, Islam T (2022) Morphomolecular and cultural characteristics and host range of \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e causing stem canker disease in drag\u0026oacute;n fruit. J Basic Microbiol 62:689\u0026ndash;700\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoutinho IBL, Freire FCO, Lima CS, Lima JS, Gon\u0026ccedil;alves FJT, Machado AR, Silva AMS, Cardoso JE (2017) Diversity of genus \u003cem\u003eLasiodiplodia\u003c/em\u003e associated with perennial tropical fruit plants in northeastern Brazil. Plant Pathol 66:90\u0026ndash;104\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDianda OZ, Issa W, Ou\u0026eacute;draogo L, Sankara P, Tollenaere C, Del Ponte EM, Fernandez D (2023) \u003cem\u003eLasiodiplodia\u003c/em\u003e species associated with mango (\u003cem\u003eMangifera indica\u003c/em\u003e L.) decline in Burkina Faso and influence of climatic factors on the disease distribution. Physiol Mol Plant Pathol 126:1020412023\u0026ndash;1020412003\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDissanayake AJ, Phillips AJL, Li XH, Hyde KD (2016) \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e: Current status of genera and species. Mycosphere 7:1001\u0026ndash;1073\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong W, Doilom M, Hyde KD, Phillips A, Yan K, To-anun C, Xu JC, Zhang H, Nalumpang S (2020) Pathogenicity of five \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e species isolated from \u003cem\u003eTectona grandis\u003c/em\u003e (teak): the pathogenic potential of \u003cem\u003eLasiodiplodia\u003c/em\u003e species. Asian J Mycol 3:399\u0026ndash;407\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Ganainy SM, Ismail AM, Iqbal Z, Elshewy ES, Alhudaib KA, Almaghasla MI, Magist\u0026agrave; D (2022) Diversity among \u003cem\u003eLasiodiplodia\u003c/em\u003e Species Causing Dieback, Root Rot and Leaf Spot on Fruit Trees in Egypt, and a Description of \u003cem\u003eLasiodiplodia newvalleyensis\u003c/em\u003e sp. nov. J Fungi 8:1203\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGunamalai L, Duanis-Assaf D, Maurer D, Sharir T, Feigenberg O, Sela N, Alkan N (2023) Comparative characterization of virulent and less virulent \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e isolates. Mol Plant Microbe Interact 36:502\u0026ndash;515\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHern\u0026aacute;ndez D, Garc\u0026iacute;a-P\u0026eacute;rez O, Perera S, Gonz\u0026aacute;lez-Carracedo MA, Rodr\u0026iacute;guez-P\u0026eacute;rez A, Siverio F (2023) Fungal pathogens associated with aerial symptoms of avocado (\u003cem\u003ePersea americana\u003c/em\u003e Mill.) in Tenerife (Canary Islands, Spain) focused on species of the family \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e. Microorganisms 11:585\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKwon JH, Choi O, Kang B, Lee Y, Park J, Kang DW, Han I, Kim J (2017) Identification of \u003cem\u003eLasiodiplodia pseudotheobromae\u003c/em\u003e causing mango dieback in Korea. Can J Plant Pathol 39:241\u0026ndash;245\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong Q, Xing S, Xie K, Jia Z, Li Z, Liu Y, Xu C, Shi X, Wang C, Yuan S (2023) First report of \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e causing stem brown rot of loquat in China. Plant Dis 107:2234\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePolashock JJ, Kramer M (2006) Resistance of blueberry cultivars to \u003cem\u003eBotryosphaeria\u003c/em\u003e stem blight and \u003cem\u003ePhomopsis\u003c/em\u003e twig blight. Hort Sci 41:1457\u0026ndash;1461\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePunithalingam E (1976) \u003cem\u003eBotryodiplodia theobromae\u003c/em\u003e. CMI Descriptions of Fungi and Bacteria: 52, Sheet-519. Commonwealt Mycological Institute, Kew, Surrey, England\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRangel-Montoya EA, Paolinelli M, Rolshausen PE, Valenzuela-Solano C, Hernandez-Martinez R (2021) Characterization of \u003cem\u003eLasiodiplodia\u003c/em\u003e species associated with grapevines in Mexico. Phytopathologia Mediterranea 60:237\u0026ndash;251\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRam\u0026iacute;rez-Gil JG, Henao-Rojas JC, Morales-Osorio JG (2021) Postharvest diseases and disorders in avocado cv. Hass and their relationship to preharvest management practices. Heliyon 7:e05905\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-G\u0026aacute;lvez E (2021) Enfermedades de la madera en los cultivos de Mango, Palto, Vid y Ar\u0026aacute;ndano, en Piura- Per\u0026uacute;. Sintomatolog\u0026iacute;a y Etiolog\u0026iacute;a. Editorial Letra. 200pp\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-G\u0026aacute;lvez E, Guerrero P, Barradas C, Crous PW, Alves A (2017) Phylogeny and pathogenicity of \u003cem\u003eLasiodiplodia\u003c/em\u003e species associated with dieback of mango in Peru. Fungal Biology 121:452\u0026ndash;465\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-G\u0026aacute;lvez E, Hil\u0026aacute;rio S, Batista E, Lopes A, Alves A (2021) \u003cem\u003eLasiodiplodia\u003c/em\u003e species associated with dieback of avocado in the coastal area of Peru. Eur J Plant Pathol 161:219\u0026ndash;232\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-G\u0026aacute;lvez E, Hil\u0026aacute;rio S, Lopes A, Alves A (2020) Diversity and pathogenicity of \u003cem\u003eLasiodiplodia\u003c/em\u003e and \u003cem\u003eNeopestalotiopsis\u003c/em\u003e species associated with stem blight and dieback of blueberry plants in Peru. Eur J Plant Pathol 157:89\u0026ndash;102\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodr\u0026iacute;guez-G\u0026aacute;lvez E, Maldonado E, Alves A (2015) Identification and pathogenicity of \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e causing dieback of table grapes in Peru. Eur J Plant Pathol 141:477\u0026ndash;489\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStatista Research Department \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://es.statista.com/acercadenosotros/nuestro-compromiso-con-la-calidad\u003c/span\u003e\u003cspan address=\"https://es.statista.com/acercadenosotros/nuestro-compromiso-con-la-calidad\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Uacute;rbez-Torres JR, Gubler WD (2009) Pathogenicity of \u003cem\u003eBotryosphaeriaceae\u003c/em\u003e species isolated from grapevine cankers in California. Plant Dis 93:584\u0026ndash;592\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Uacute;rbez-Torres JR, Leavitt GM, Guerrero JC, Guevara J, Gubler WD (2008) Identification and pathogenicity of \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e and \u003cem\u003eDiplodia seriata\u003c/em\u003e, the causal agents of bot canker disease of grapevines in Mexico. Plant Dis 92:519\u0026ndash;529\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang W, Groenewald JZ, Lombard L, Schumacher RK, Phillips AJL, Crous PW (2021) Evaluating species in \u003cem\u003eBotryosphaeriales\u003c/em\u003e. Persoonia \u0026ndash; Mol Phylogeny Evol Fungi 46:63\u0026ndash;115\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"tropical-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tppa","sideBox":"Learn more about [Tropical Plant Pathology](https://www.springer.com/journal/40858)","snPcode":"40858","submissionUrl":"https://www.editorialmanager.com/tppa","title":"Tropical Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Avocado, external and internal necroses, Lasiodiplodia theobromae, Persea americana, symptom development, virulence","lastPublishedDoi":"10.21203/rs.3.rs-4496463/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4496463/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eLasiodiplodia theobromae\u003c/em\u003e is a pathogenic fungus of more than 500 plant species, including avocado (\u003cem\u003ePersea americana\u003c/em\u003e Mill.). The global production volume of avocado exceeded 911,000 metric tons in 2023 (Statista, 2024). Although detailed quantitative surveys of yield losses caused by \u003cem\u003eL. theobromae\u003c/em\u003e are not available for most avocado-producing regions, estimates indicate that this pathogen is associated with stem-end rot disease in 30\u0026ndash;35% of plots located in the Department of Antioquia in Colombia (Ram\u0026iacute;rez-Gil et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), suggesting an enormous economic impact. As yield losses strongly depend on the virulence of isolates, a reliable method for determining their aggressiveness is indispensable for initiating disease control measures. In this study, we compared progression of external and internal necrosis caused by the aggressive \u003cem\u003eL. theobromae\u003c/em\u003e isolate LA-VLCA3 inoculated into wounded middle parts of the stem and onto excised apices. Irrespective of the inoculation method, internal progression of necroses preceeded that of external necroses. Spreading of external and internal necroses was significantly more severe in plants inoculated at the apex than in mid-stem inoculated plants. We conclude that apex inoculation causes stronger symptoms and is therefore better suited to compare virulence of isolates than mid-stem inoculation.\u003c/p\u003e","manuscriptTitle":"Lasiodiplodia theobromae disease symptom development in young avocado (Persea americana L.) plants depends on the inoculation method","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-12 09:16:34","doi":"10.21203/rs.3.rs-4496463/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-07-20T10:09:51+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-17T17:18:13+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-09T05:53:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Tropical Plant Pathology","date":"2024-07-02T09:32:31+00:00","index":"","fulltext":""},{"type":"decision","content":"Major revisions","date":"2024-06-27T14:34:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"tropical-plant-pathology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"tppa","sideBox":"Learn more about [Tropical Plant Pathology](https://www.springer.com/journal/40858)","snPcode":"40858","submissionUrl":"https://www.editorialmanager.com/tppa","title":"Tropical Plant Pathology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e0c2bf9e-3bac-463e-b00f-ed7838ca806d","owner":[],"postedDate":"August 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-11-04T20:27:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-12 09:16:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4496463","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4496463","identity":"rs-4496463","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}