Morphocultural Characterization and Aggressiveness of Fusarium stilboides Isolates from Four Coffee Growing Regions in Tanzania

Morphocultural Characterization and Aggressiveness of Fusarium stilboides Isolates from Four Coffee Growing Regions in Tanzania | 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 Research Article Morphocultural Characterization and Aggressiveness of Fusarium stilboides Isolates from Four Coffee Growing Regions in Tanzania Neyonkulu Sedikia Kahishai, Nuhu Aman Mbwebwe, Deusdedit Kilambo, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6807950/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fusarium stilboides , the causative agent of Fusarium bark disease (FBD) in Coffea arabica , poses a significant threat to coffee production. Despite its impact, limited research has explored the pathogen’s morphological diversity and pathogenic variability in Tanzania. This study aimed to characterize the morphocultural traits and aggressiveness of F. stilboides isolates collected from four coffee-growing regions in Tanzania: Suji (Same District- Kilimanjaro region), Mahande and Mbangamao (Mbinga DC- Ruvuma region), Ayalabe (Karatu District- Arusha), and Bara (Mbozi District- Songwe region). Five isolates were obtained from symptomatic coffee trees. They were then cultured on Potato Dextrose Agar (PDA), and assessed for colony morphology, pigmentation, growth rate, and sporulation. Evaluation of pathogenicity was through stem injection and soil-drenching methods of inoculation on C. arabica seedlings, with disease severity monitored over 112 days. Results revealed significant variations in colony color, texture, and growth rate among isolates. Sporulation capacity also differed significantly, with isolates KSS24 and SVB24 exhibiting abundant sporulation, while RMM24 had the lowest. Pathogenicity assays showed a significant difference (P < 0.05). Isolate KSS24 was the most aggressive, reaching a mean disease severity score of 3.00 by 63 days after inoculation, whereas AKA24 exhibited the lowest severity (0.67). These findings highlight the morphological and pathogenic variability of F. stilboides in Tanzania, underscoring the need for further studies on molecular characterization and environmental interactions influencing pathogenicity, which could further inform breeding programs aimed at developing resistant coffee varieties. Fusarium Bark Disease Fusarium stilboides Coffea arabica Morphocultural characterization Figures Figure 1 Figure 2 Introduction Fusarium bark disease (FBD), caused by Fusarium stilboides , is reemerging to be one of the major constraint to Coffea arabica production, leading to severe yield losses (Gathuru et al. , 2021). The Fusarium genus comprises both pathogenic and non-pathogenic strains commonly found in soils. The pathogenic strains significantly affect various economically important crops, leading to plant wilt and root infections (Bertrand et al. , 2000).To date, over 120 formae speciales ( ff. spp .) have been identified, each infecting only one or a few specific host plant species (Oliveira et al. , 2021 ). Most Coffea arabica cultivars are vulnerable to Fusarium species , which cause tree wilt and dry root rot (Al-Faifi et al. , 2022). Fusarium Bark disease manifests as bark lesions, necrosis, and stem girdling, ultimately reducing plant vigor and productivity (Waller et al. , 2007). Fusarium species have been widely reported as significant pathogens of coffee, affecting various plant parts and leading to economic losses (Serani, Taligoola, and Hakiza, 2007). Infected trees often exhibit external stem discoloration, vascular necrosis, and dieback symptoms that are intensified by environmental conditions favoring fungal growth and infection (Siddiqi and Corbett, 1968). In recent years, many coffee farmers in coffee-growing regions have complained about rapid wilting and death of their coffee trees without knowing the cause. Interestingly, it has been discovered that the symptoms observed are associated with Fusarium species and Fusarium stilboides being the major cause in Coffea arabica (Al-Faifi et al. , 2022). Despite its impact, limited research has explored the morphocultural diversity and pathogenic variability of F. stilboides isolates in Tanzania.Therefore, Understanding the pathogen’s morphological diversity and aggressiveness is crucial for developing effective management strategies, including resistant coffee varieties and targeted disease control methods (Ploetz, 2016). Studies on Fusarium species have shown that colony morphology, pigmentation, and sporulation capacity are linked to pathogenic potential and adaptability (Lazarotto et al. , 2014). Morphocultural variation is also associated with genetic diversity and host specificity in Fusarium populations affecting perennial crops (Tshilenge, Kalonji, and Tshilenge, 2010). However, little is known about the morphocultural traits of F. stilboides isolates in Tanzania. This study aimed to characterize the morphocultural traits of F. stilboides isolates from five coffee growing regions and evaluate their aggressiveness in a controlled experiment. Materials and Methods Isolation and Morphocultural Evaluation of F. stilboides Samples were collected from four C. arabica growing regions, one location in each region with an exception of Ruvuma region where two villages were selected basing on FBD prevalence: Suji (Same District- Kilimanjaro region), Mahande and Mbangamao (Mbinga DC- Ruvuma region), Ayalabe (Karatu District- Arusha region), and Bara (Mbozi District- Songwe region). Infected coffee trees ( Coffea arabica ) exhibiting typical Fusarium bark disease (FBD) symptoms, including leaf yellowing, wilting, and bark lesions, were selected for sampling. Bark tissues from symptomatic trees were carefully excised using a sterile scalpel and stored in sterile paper bags for transportation to the laboratory. Samples were processed within 24 hours to prevent contamination and deterioration. Surface Sterilization and Fungal Isolation Collected bark tissues were cut into small segments (approximately 5 x 5 mm) and surface-sterilized by immersing them in 70% ethanol for 1 minute, followed by 1% sodium hypochlorite (NaOCl) solution for 3 minutes, and finally rinsed three times in sterile distilled water. The surface-sterilized segments were dried on sterile filter paper under laminar airflow before plating. Media Preparation and Fungal Culturing Potato Dextrose Agar (PDA) was prepared by dissolving 39 g of PDA powder in 1 L of distilled water, followed by autoclaving at 121°C for 15 minutes. After cooling to approximately 50°C, the medium was poured into sterile Petri dishes (90 mm in diameter) under aseptic conditions and left to solidify.Sterilized bark segments were placed onto PDA plates and incubated at 25 ± 2°C under a 12-hour light/dark cycle for 5 to 7 days. Emerging fungal colonies were sub-cultured onto fresh PDA plates to obtain pure cultures. Colony Morphology Characterization Morphological characteristics, including colony color, texture, margin type, and growth rate, were observed, measured (in cm) and recorded daily for seven days. Colony pigmentation was assessed from both the upper and reverse sides of the plates. Mycelial texture was categorized as cottony, powdery, or velvety, while edge types were classified as smooth, irregular, or filamentous. Pathogenicity Assessment in Coffee Seedlings Confirmed isolates from the Tanzania Coffee Research Institute Plant Pathology Laboratory were used. The isolates were cultured on PDA for 7 days before inoculum preparation. The treatments included five F. stilboides isolates: Suji (KSS24), Mahande (RMM24), Mbangamao (RMMb24), Ayalabe (AKA24), and Bara (SVB24). Inoculation was performed on true-to-type N-39 Coffea arabica seedlings. Inoculum Preparation and Quantification F. stilboides cultures grown on PDA for seven days were cut using sterile blades and transferred to a 500 mL beaker. Cultures from 15 Petri dishes were combined, and 150 mL of sterile distilled water with 0.1% Tween-80 was added. The mixture was shaken for 10 minutes on an electric shaker to release spores and filtered through a sterile muslin cloth. A 10 µL aliquot of the suspension was transferred to a hemocytometer, and spores were counted in five squares per chamber. Dilutions were performed using C₁V₁ = C₂V₂ formula to adjust concentrations to 10⁵ and 10⁶ spores/mL. Pathogen Inoculation Two inoculation methods were tested: stem injection and soil drenching. The stem injection method followed Li et al. (2021), where a sterile disposable needle created a hole, and 1 µL of F. stilboides spore solution was injected. For the soil drenching method, roots were slightly injured before applying 10 mL of the spore solution to the soil surface. Control seedlings received sterile distilled water. The seedlings were maintained at room temperature for two weeks before being moved to a net house, arranged in a complete randomized design, and managed under standard agronomic practices. Pathogen Confirmation To confirm infection, F. stilboides was re-isolated from symptomatic seedlings. Seedlings were uprooted, washed, and longitudinally sectioned at the inoculation site. Tissue samples were surface-sterilized (2% sodium hypochlorite for 1 min, 70% ethanol for 1 min, and rinsed four times in sterile distilled water) and blot-dried. Five pieces of tissue sample per treatment were plated on PDA, incubated at 24°C, and monitored for F. stilboides growth. Microscopic Examination of Fusarium Isolates Spore Spore concentration was determined using a Neubauer hemocytometer under a light microscope (×40 magnification). A 10 µL aliquot of the spore suspension was loaded into the hemocytometer chamber. Spores were counted in five squares, and the spore concentration (spores/mL) was calculated.To assess the morphology of Fusarium spores, 10 µL of the spore suspension was placed on a clean glass slide, stained with lactophenol cotton blue, and covered with a sterile cover slip. Observations were conducted under a light microscope. Spore characteristics including shape and size were documented. Voucher Specimen and Identification Plant material was collected in four coffee-growing regions in Tanzania: Suji (Same District- Kilimanjaro region), Mahande and Mbangamao (Mbinga DC- Ruvuma region), Ayalabe (Karatu District- Arusha), and Bara (Mbozi District- Songwe region). Neyonkulu Sedikia Kahishai, Nuhu Aman Mbwebwe, Deusdedit Kilambo, and Fatuma Jumapili Ramadhani identified the specimen, and vouchers were deposited at the Tanzania Coffee Research Institute Herbarium. Data Collection and Analysis Morphocultural data were recorded based on colony morphology, growth rate, pigmentation, and sporulation. Spore density was quantified using a hemocytometer (Leslie and Summerell, 2006). Disease severity was assessed using a modified 0–3 scale: 0 = Symptomless plants, 1 = < 10% limited wilting, 2 = 10–30% severe yellowing and wilting, 3 = 30–50% very severe yellowing and wilting. Disease incidence was calculated using the formula by Cooke et al. (2006). Data were subjected to ANOVA in GENSTAT 2014 16th edition software to generate means, variances and standard error of difference. The means were separated using Tukey’s Honest Significant Difference test at p < 0.05. Results and discussions Sporulation and Conidial Morphology of Fusarium stilboides Isolates The five Fusarium stilboides isolates exhibited notable variation in sporulation capacity and conidial morphology. Among the isolates, KSS24 and SVB24 demonstrated abundant sporulation, whereas AKA24 exhibited moderate sporulation. In contrast, RMMb24 and RMM24 showed sparse sporulation under the given conditions (Table 1 ). Microscopic examination revealed that four isolates—KSS24, SVB24, AKA24, and RMMb24 produced round microconidia, which are small, single-celled, and non-septate structures typical of certain Fusarium species during specific developmental stages (Leslie and Summerell, 2006). The presence of microconidia in these isolates suggests an adaptation to environmental or physiological conditions favoring their production. On the other hand, the RMM24 isolate, despite its sparse sporulation, produced canoe-shaped, septate macroconidia, a characteristic feature of many Fusarium species . High-magnification microscopy confirmed the distinct elongated shape and segmentation of these macroconidia. Table 1 Sporulation Levels and Conidial Morphology of Fusarium stilboides Isolates Isolate Sporulation Level (spore/ml) Conidial Shape RMM24 Sparse (3.0x10 6 ) Canoe-shaped macroconidia KSS24 Abundant(3.6x10 6 ) Round microconidia AKA24 Moderate (2.26x10 7 ) Round microconidia SVB24 Abundant (5.6x10 7 ) Round microconidia RMMb24 Moderate (1.08x10 7 ) Round microconidia The observed variation in sporulation among Fusarium stilboides isolates aligns with previous studies highlighting morphocultural diversity within Fusarium species . (Leslie and Summerell, 2006; Aoki et al. , 2014; Lazarotto et al. , 2014). The high sporulation levels observed in KSS24 and SVB24 suggest a potential advantage in dissemination and infection, as conidial density is a critical factor in pathogenicity (Cai et al. , 2022). The tendency of the RMM24 isolate to produce macroconidia instead of microconidia under laboratory conditions may indicate the influence of specific environmental or physiological factors on sporulation, a pattern also reported in other Fusarium species (Ortoneda et al. , 2004; Tshilenge et al. , 2010). Variations in sporulation patterns and conidial morphology can significantly influence the pathogenicity and epidemiology of Fusarium stilboides . Isolates producing higher quantities of microconidia, such as KSS24 and SVB24, may exhibit enhanced potential for rapid disease spread, as microconidia are often more easily dispersed (Summerell, 2019; Serani et al. , 2007). In many Fusarium species , microconidia serve as primary inoculum, facilitating both initial infection and secondary spread under favorable environmental conditions. The RMM24 isolate, which produces canoe-shaped macroconidia, may have a lower potential for immediate infection and rapid disease spread. Macroconidia, being generally larger, often require specific conditions for germination, potentially resulting in a slower infection process compared to microconidia. However, their presence could indicate a more aggressive or persistent infection strategy, as macroconidia frequently function as survival structures, enabling the pathogen to endure unfavorable environmental conditions (Ploetz, 2016). Differences in sporulation among isolates may also reflect underlying genetic variability associated with geographic origin. Studies on Fusarium species infecting coffee, such as F. xylarioides and F. stilboides , have demonstrated that environmental factors including temperature, humidity, and media composition can influence sporulation rates and conidial morphology (Acuña et al. , 2021; Gathuru et al. , 2021). The findings of this study suggest similar variability among Tanzanian F. stilboides isolates, with potential implications for disease epidemiology and management strategies. Growth Rate and Morphological Characteristics of Fusarium stilboides Isolates The growth rate of Fusarium stilboides isolates over seven days, illustrated in Fig. 1 , further emphasizes their variability. SVB24 exhibited the highest growth rate, reaching the greatest colony diameter by Day 7, followed by RMMb24, RMM24 and KSS24, while AKA24 displayed the slowest expansion. There was a significant difference in growth rate (P < 0.05) among the isolates over time.These differences in growth rates may be linked to sporulation capacity and conidial morphology suggesting that isolates with abundant microconidia such as SVB24 have enhanced colonization potential. Morphological Study of F. stilboides Isolates Morphological characteristics of F. stilboides isolates (Table 2 ) indicate notable variation in colony color, texture, and pigmentation (Fig. 2 ). Isolates with higher growth rates, such as SVB24 and RMMb24, were predominantly cottony or slimy in texture with white or pink color. In contrast, the slow-growing isolate AKA24 exhibited a cream-colored cottony colony, potentially indicative of lower adaptability to laboratory conditions. Differences in sporulation, growth rates, and colony morphology highlight the adaptive strategies of F.stilboides isolates under varying environmental conditions. Previous studies on Fusarium species have demonstrated that pigmentation, texture, and edge type may be linked to pathogenic fitness, resistance to environmental stress, and virulence (Acuña et al. , 2021). Thus, these findings emphasize the need for further studies to explore the genetic and environmental factors influencing these traits. Table 2 Fusarium Stilboides cultures Morphological Characteristics Isolate Colony color pigmentation Colony texture Edge type KSS24 White Pink Cottony Smooth RMM24 Black Purple Velvety Wavy RMMb24 Pink Pink Slimy Smooth SVB24 White Pink Cottony Smooth AKA24 Cream Pink Cottony Wavy Virulence of F. stilboides Isolates The severity of F. stilboides isolates varied significantly across the different treatments over period (Table 3 ). The distilled water control exhibited no symptoms throughout the experimental period. On the other hand, the isolate KSS24 exhibited the highest disease severity progression, reaching a mean score of 3.00 from 63 DAI onwards. Table 3 Disease Severity Progression of Fusarium Stilboides Isolates over Time Isolate code 28DAI 35DAI 42DAI 49DAI 56DAI 63DAI 70DAI 77DAI 84DAI 91DAI 98DAI 105DAI 112DAI AKA24 0.00 a 0.67 a 0.67 a 0.67 a 0.67 a 0.67 a 0.67 a 0.67 a 0.67 a 0.67 a 0.67 a 0.67 a 0.67 a Distilled water 0.00 a 0.00 a 0.00 a 0.00 a 0.00 a 0.00 a 0.00 a 0.00 a 0.00 a 0.00 a 0.00 a 0.00 a 0.00 a RMM24 0.67 a 1.00 a 2.00 a 2.00 a 2.00 a 2.00 a 2.00 ab 2.00 ab 2.00 ab 2.00 ab 2.00 ab 2.00 ab 2.00 ab RMMb24 0.67 a 1.33 a 1.33 a 1.33 a 1.33 a 1.33 a 1.33 ab 1.33 ab 1.33 ab 1.33 ab 1.33 ab 1.33 ab 1.33 ab SVB24 0.67 a 1.00 a 1.00 a 1.00 a 1.00 a 1.00 a 1.00 ab 1.00 ab 1.00 ab 1.00 ab 1.00 ab 1.00 ab 1.00 ab KSS24 1.67 a 1.67 a 2.00 a 2.00 a 2.00 a 3.00 a 3.00 b 3.00 b 3.00 b 3.00 b 3.00 b 3.00 b 3.00 b Grand mean 0.61 0.94 1.17 1.17 1.17 1.33 1.33 1.33 1.33 1.33 1.33 1.33 1.33 S.e.d 0.609 0.745 0.72 0.72 0.72 0.638 0.638 0.638 0.638 0.638 0.638 0.638 0.638 cv% 122 96.7 75.6 75.6 75.6 58.6 58.6 58.6 58.6 58.6 58.6 58.6 58.6 F prob. 0.148 0.372 0.104 0.104 0.104 0.008 0.008 0.008 0.008 0.008 0.008 0.008 0.008 *Means with the same letter in the same column indicate no significant differences (p ≤ 0.05) according to Tukey’s Honestly Significance Difference. *DAI = Days After Inoculation; cv%=coefficient of variation; F prob = F probability Among the test isolates, RMM24 showed a gradual increase in disease severity, attaining a mean score of 2.00 from 35 days after inoculation and no further disease development. Isolates RMMb24 and SVB24 exhibited relatively lower disease severity, with both reaching a maximum of 1.33 and 1.00 mean score, respectively at 112 days after inoculation. AKA24 showed the lowest disease severity among the isolates, remaining at a mean score of 0.67 throughout the experiment, comparable to the distilled water control. There were significant differences (P < 0.05) among the isolates over time. The F probability values indicated no significant differences among treatments at early evaluation stages, but significant variations emerged from 63 days after inoculation onward (p < 0.05). The coefficient of variation (CV%) decreased over time, indicating more stability in disease severity scores as the experiment progressed. The high severity recorded in KSS24 compared to other isolates indicates its higher aggressiveness, aligning with previous studies on Fusarium species (Ortoneda et al. , 2004), where pathogenic variability has been reported among isolates from different geographical regions. The relatively lower severity in RMMb24 and SVB24 suggests that these isolates may be less virulent or slower in colonization under the given experimental conditions. Conclusion and Recommendations This study highlights significant variations in the sporulation, conidial morphology, growth rate, and virulence of Fusarium stilboides isolates collected from different coffee grown regions of Tanzania. The observed differences suggest that certain isolates, such as KSS24, possess enhanced sporulation and pathogenic potential, which may contribute to increased disease spread in coffee plants. On the other hand, isolates like RMM24 and AKA24 exhibited lower sporulation and disease severity, indicating potential variability in aggressiveness and adaptability. The presence of both microconidia and macroconidia across different isolates suggests diverse survival and infection strategies, which may influence disease epidemiology and management approaches. Understanding the variability within Fusarium stilboides populations will be crucial in mitigating the impact of this pathogen on coffee production in Tanzania and beyond. The findings of this study show the importance of characterizing Fusarium isolates at both morphological and pathogenic levels to develop targeted disease management strategies. Future research should focus on molecular characterization and environmental interactions influencing pathogenicity, which could further inform breeding programs aimed at developing resistant coffee varieties. Declarations FUNDING AND CONSENT DECLARATION Tanzania Coffee Research Institute provided funding. Ethics, Consent to Participate, and Consent to Publish declarations: not applicable. Clinical trial number: not applicable. Data availability statement: The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. Voucher specimen and identification: Plant material was collected in four coffee-growing regions in Tanzania: Suji (Same District- Kilimanjaro region), Mahande and Mbangamao (Mbinga DC- Ruvuma region), Ayalabe (Karatu District- Arusha), and Bara (Mbozi District- Songwe region). Neyonkulu Sedikia Kahishai, Nuhu Aman Mbwebwe, Deusdedit Kilambo, and Fatuma Jumapili Ramadhani identified the specimen, and vouchers were deposited at the Tanzania Coffee Research Institute Herbarium. Ethics and Guidelines: No human participants or live vertebrates were involved in this study. Collection of fungal isolates followed institutional (TaCRI) and national (Tanzania) guidelines. Plant specimens were identified and voucher specimens deposited under institutional (TaCRI) policies in compliance with Tanzanian biosafety protocols. Author Contribution N.S.K and D.K wrote the main manuscript, N.A.M and F.J.R prepared some figures and all authors reviewed the manuscript. Data Availability The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. References Acuña R, Mena J, Gil L. Fusarium species infecting coffee crops: Morphological and molecular characterization. J Plant Pathol. 2021;103(2):345–57. https://doi.org/10.1007/s42161-021-00915-3. Acuña R, Smith DR, Jones TK. Environmental factors influencing Fusarium sporulation and pathogenicity in coffee crops. Plant Pathol J. 2021;37(2):215–29. Aoki T, O’Donnell K, Homma Y, Lattanzi AR. Morphological and molecular characterization of Fusarium species associated with coffee plants. Mycol Res. 2014;118(1):1–11. https://doi.org/10.1016/j.mycres.2014.01.004 . Aoki T, O’Donnell K, Geiser DM. Systematics of key Fusarium pathogens of coffee. Mycol Res. 2014;118(9):865–78. •AL-Faifi Z, Alsolami W, Abada E, Khemira H, Almalki G, Modafer Y. (2022). Fusarium oxysporum and Colletotrichum musae associated with wilt disease of Coffea arabica in coffee gardens in Saudi Arabia. International Journal of Agronomy, 2022, 3050495. https://doi.org/10.1155/2022/3050495 •Bertrand B, Nunez C, Sarah J-L. Disease complex in coffee involving Meloidogyne arabicida and Fusarium oxysporum . Plant Pathol. 2000;49(3):383–8. https://doi.org/10.1046/j.1365-3059.2000.00456.x . Cai F, Gao R, Zhao Z, Ding M, Jiang S, Zhao L. Conidial density and pathogenic potential of Fusarium species. Fungal Biology. 2022;126(4):324–36. Cai X, Wang Y, Li H, Zhang Y. Conidial density and pathogenicity in Fusarium species: A comparative study. J Plant Pathol. 2022;104(2):455–68. Cooke BM, Jones DG, Kaye B. Diseases of Field Crops. CRC; 2006. Gathuru EM, Mwangi LM, Otieno N. Pathogenic variability in Fusarium spp. causing coffee diseases. Afr J Plant Sci. 2021;15(3):87–96. Gathuru EM, Omondi CO, Nyabundi JO. Occurrence and management of Fusarium bark disease in Kenya. J Coffee Res. 2021;50(2):89–102. Lazarotto M, Milanesi PM, Muniz MFB, Reiniger LRS, Beltrame R, Harakava R, Blume E. Morphological and molecular characterization of Fusarium spp. pathogenic to pecan tree in Brazil. Genet Mol Res. 2014;13(4):9390–402. https://doi.org/10.4238/2014.November.18.13 . Leslie JF, Summerell BA. The Fusarium Laboratory Manual. Blackwell Publishing; 2006. Li X, Zhang Y, Wang J, Chen H. Development of an inoculation technique for rapidly evaluating maize inbred lines for resistance to Fusarium spp. Plant Dis. 2021;105(12):3683–9. https://doi.org/10.1094/PDIS-09-20-2016-SR . •Oliveira L, de Rodrigues JMG, Silva AAC, Oliveira EKCe, de Barros ACS, Fraga MC, de Nascimento E O., and, Silva MRM. (2021). Morphological and phylogenetic characterization of Fusarium Link . Australian Journal of Crop Science, 15 (12), Article p3292. https://doi.org/10.21475/ajcs.21.15.12.p3292 Ortoneda M, Guarro J, Madrid MP, Caracuel Z, Roncero MIG, Mayayo E, Di Pietro A. Fusarium pathogenicity and sporulation under different environmental conditions. Appl Environ Microbiol. 2004;70(5):3206–12. Ploetz RC. Fusarium species associated with coffee diseases: Taxonomy and control. Phytopathology. 2016;106(2):171–87. https://doi.org/10.1094/PHYTO-09-15-0230-RVW . Serani S, Taligoola HK, Hakiza GJ. An investigation into Fusarium spp. associated with coffee and banana plants as potential pathogens of robusta coffee. Afr J Ecol. 2007;45(1):91–5. https://doi.org/10.1111/j.1365-2028.2007.00865.x . Siddiqi MA, Corbett DCM. Coffee bark diseases in Malawi: Properties of the causal organism and conditions favoring the disease. Trans Br Mycological Soc. 1968;51(1):129–35. https://doi.org/10.1016/S0007-1536(68)80017-8 . Summerell BA. Fusarium : Identification and diversity in agricultural and natural ecosystems. Phytopathology. 2019;109(4):512–26. https://doi.org/10.1094/PHYTO-01-19-0030-R . Summerell BA. Fifty years of Fusarium : How understanding the ecology of Fusarium species has informed plant pathology. Fungal Biology Reviews. 2019;33(1):20–30. Tshilenge L, Kalonji A, Tshilenge PJ. Determination of cultural and biometrical characters of Fusarium species isolated from plant material harvested from coffee ( Coffea canephora Pierre) infected with coffee wilt disease in the Democratic Republic of Congo. Afr J Agric Res. 2010;5(22):3145–50. Waller JM, Bigger M, Hillocks RJ. Coffee Pests, Diseases, and Their Management. CABI Publishing; 2007. Yang J, Han J, Jing Y, Li S, Lan B, Zhang Q, Yin K. Virulent Fusarium isolates with diverse morphologies show similar invasion and colonization strategies in alfalfa. Front Plant Sci. 2024;15:1390069. https://doi.org/10.3389/fpls.2024.1390069 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. 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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-6807950","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":484877302,"identity":"2c175f47-7b53-4007-be56-a17fc12c0ba8","order_by":0,"name":"Neyonkulu Sedikia Kahishai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYJACxgaGAzwMEgyMD4AcHj5StDAbgLSwEauFAaiFTQLEI6jFnL3H8OGMmjsy8rObn1V+zbGTYWNgfvjoBh4tlj1njA03HHvGY3DnmNlt2W3JQIexGRvn4NFicCPHTPIB22EeA4kEs9uS25iBWnjYpPFquf8GqOXfYR75GenfiiW31ROh5QaPmeTGtsM8DEDrGD9uO0yEljNpxYYz+4AOu5FTLM247TgPGzMhvxw/vPFhz7fD9kCHbfz4c1u1PT9788PH+LQwMHAYwJnMPGASr3IQYH8AZzL+IKh6FIyCUTAKRiIAACXlSj6X50C+AAAAAElFTkSuQmCC","orcid":"","institution":"Tanzania Cofee Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Neyonkulu","middleName":"Sedikia","lastName":"Kahishai","suffix":""},{"id":484877303,"identity":"56144d49-0c1e-4967-97e8-40ee29ec4e20","order_by":1,"name":"Nuhu Aman Mbwebwe","email":"","orcid":"","institution":"Tanzania Cofee Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Nuhu","middleName":"Aman","lastName":"Mbwebwe","suffix":""},{"id":484877304,"identity":"c4224bb5-51a6-4308-b952-192229e8827b","order_by":2,"name":"Deusdedit Kilambo","email":"","orcid":"","institution":"Tanzania Cofee Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Deusdedit","middleName":"","lastName":"Kilambo","suffix":""},{"id":484877305,"identity":"c01ecc4c-9876-4ea9-b4d5-11d1fec8ff96","order_by":3,"name":"Fatuma Jumapili Ramadhani","email":"","orcid":"","institution":"Tanzania Cofee Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Fatuma","middleName":"Jumapili","lastName":"Ramadhani","suffix":""}],"badges":[],"createdAt":"2025-06-03 07:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6807950/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6807950/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87302943,"identity":"e62e7ea0-bf3e-40b2-9c6e-77b3fdf88d02","added_by":"auto","created_at":"2025-07-22 13:40:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":48537,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGrowth Rate of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eFusarium Stilboides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Isolates\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6807950/v1/1680f68fc4a94d0f0adf33fe.png"},{"id":87302946,"identity":"2feb033b-7d8f-4b19-bf2d-140312fe7581","added_by":"auto","created_at":"2025-07-22 13:40:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":348995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eColonies of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eF. Stilboides\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e after 7 days of incubation on PDA media\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6807950/v1/479aabed0c835dea9e40cafc.png"},{"id":97249410,"identity":"2f468653-59c6-4501-97bf-73126a855a7b","added_by":"auto","created_at":"2025-12-02 13:12:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1626271,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6807950/v1/509cbc47-6bef-4f30-aedd-8c8da4871cb5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Morphocultural Characterization and Aggressiveness of Fusarium stilboides Isolates from Four Coffee Growing Regions in Tanzania","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFusarium bark disease (FBD), caused by \u003cem\u003eFusarium stilboides\u003c/em\u003e, is reemerging to be one of the major constraint to \u003cem\u003eCoffea arabica\u003c/em\u003e production, leading to severe yield losses (Gathuru \u003cem\u003eet al.\u003c/em\u003e, 2021). The Fusarium genus comprises both pathogenic and non-pathogenic strains commonly found in soils. The pathogenic strains significantly affect various economically important crops, leading to plant wilt and root infections (Bertrand \u003cem\u003eet al.\u003c/em\u003e, 2000).To date, over 120 \u003cem\u003eformae speciales\u003c/em\u003e (\u003cem\u003eff. spp\u003c/em\u003e.) have been identified, each infecting only one or a few specific host plant species (Oliveira \u003cem\u003eet al.\u003c/em\u003e, 2021 ). Most \u003cem\u003eCoffea arabica\u003c/em\u003e cultivars are vulnerable to \u003cem\u003eFusarium species\u003c/em\u003e, which cause tree wilt and dry root rot (Al-Faifi \u003cem\u003eet al.\u003c/em\u003e, 2022). Fusarium Bark disease manifests as bark lesions, necrosis, and stem girdling, ultimately reducing plant vigor and productivity (Waller \u003cem\u003eet al.\u003c/em\u003e, 2007). \u003cem\u003eFusarium species\u003c/em\u003e have been widely reported as significant pathogens of coffee, affecting various plant parts and leading to economic losses (Serani, Taligoola, and Hakiza, 2007). Infected trees often exhibit external stem discoloration, vascular necrosis, and dieback symptoms that are intensified by environmental conditions favoring fungal growth and infection (Siddiqi and Corbett, 1968). In recent years, many coffee farmers in coffee-growing regions have complained about rapid wilting and death of their coffee trees without knowing the cause. Interestingly, it has been discovered that the symptoms observed are associated with \u003cem\u003eFusarium species\u003c/em\u003e and \u003cem\u003eFusarium stilboides\u003c/em\u003e being the major cause in \u003cem\u003eCoffea arabica\u003c/em\u003e (Al-Faifi \u003cem\u003eet al.\u003c/em\u003e, 2022). Despite its impact, limited research has explored the morphocultural diversity and pathogenic variability of \u003cem\u003eF. stilboides\u003c/em\u003e isolates in Tanzania.Therefore, Understanding the pathogen\u0026rsquo;s morphological diversity and aggressiveness is crucial for developing effective management strategies, including resistant coffee varieties and targeted disease control methods (Ploetz, 2016).\u003c/p\u003e\u003cp\u003eStudies on \u003cem\u003eFusarium species\u003c/em\u003e have shown that colony morphology, pigmentation, and sporulation capacity are linked to pathogenic potential and adaptability (Lazarotto \u003cem\u003eet al.\u003c/em\u003e, 2014). Morphocultural variation is also associated with genetic diversity and host specificity in Fusarium populations affecting perennial crops (Tshilenge, Kalonji, and Tshilenge, 2010). However, little is known about the morphocultural traits of \u003cem\u003eF. stilboides\u003c/em\u003e isolates in Tanzania. This study aimed to characterize the morphocultural traits of \u003cem\u003eF. stilboides\u003c/em\u003e isolates from five coffee growing regions and evaluate their aggressiveness in a controlled experiment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eIsolation and Morphocultural Evaluation of\u003c/b\u003e \u003cb\u003eF. stilboides\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSamples were collected from four \u003cem\u003eC. arabica\u003c/em\u003e growing regions, one location in each region with an exception of Ruvuma region where two villages were selected basing on FBD prevalence: Suji (Same District- Kilimanjaro region), Mahande and Mbangamao (Mbinga DC- Ruvuma region), Ayalabe (Karatu District- Arusha region), and Bara (Mbozi District- Songwe region).\u003c/p\u003e\u003cp\u003eInfected coffee trees (\u003cem\u003eCoffea arabica\u003c/em\u003e) exhibiting typical Fusarium bark disease (FBD) symptoms, including leaf yellowing, wilting, and bark lesions, were selected for sampling. Bark tissues from symptomatic trees were carefully excised using a sterile scalpel and stored in sterile paper bags for transportation to the laboratory. Samples were processed within 24 hours to prevent contamination and deterioration.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSurface Sterilization and Fungal Isolation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eCollected bark tissues were cut into small segments (approximately 5 x 5 mm) and surface-sterilized by immersing them in 70% ethanol for 1 minute, followed by 1% sodium hypochlorite (NaOCl) solution for 3 minutes, and finally rinsed three times in sterile distilled water. The surface-sterilized segments were dried on sterile filter paper under laminar airflow before plating.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMedia Preparation and Fungal Culturing\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePotato Dextrose Agar (PDA) was prepared by dissolving 39 g of PDA powder in 1 L of distilled water, followed by autoclaving at 121\u0026deg;C for 15 minutes. After cooling to approximately 50\u0026deg;C, the medium was poured into sterile Petri dishes (90 mm in diameter) under aseptic conditions and left to solidify.Sterilized bark segments were placed onto PDA plates and incubated at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under a 12-hour light/dark cycle for 5 to 7 days. Emerging fungal colonies were sub-cultured onto fresh PDA plates to obtain pure cultures.\u003c/p\u003e\u003cp\u003e\u003cb\u003eColony Morphology Characterization\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMorphological characteristics, including colony color, texture, margin type, and growth rate, were observed, measured (in cm) and recorded daily for seven days. Colony pigmentation was assessed from both the upper and reverse sides of the plates. Mycelial texture was categorized as cottony, powdery, or velvety, while edge types were classified as smooth, irregular, or filamentous.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePathogenicity Assessment in Coffee Seedlings\u003c/b\u003e\u003c/p\u003e\u003cp\u003eConfirmed isolates from the Tanzania Coffee Research Institute Plant Pathology Laboratory were used. The isolates were cultured on PDA for 7 days before inoculum preparation. The treatments included five \u003cem\u003eF. stilboides\u003c/em\u003e isolates: Suji (KSS24), Mahande (RMM24), Mbangamao (RMMb24), Ayalabe (AKA24), and Bara (SVB24). Inoculation was performed on true-to-type N-39 \u003cem\u003eCoffea arabica\u003c/em\u003e seedlings.\u003c/p\u003e\u003cp\u003e\u003cb\u003eInoculum Preparation and Quantification\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eF. stilboides\u003c/em\u003e cultures grown on PDA for seven days were cut using sterile blades and transferred to a 500 mL beaker. Cultures from 15 Petri dishes were combined, and 150 mL of sterile distilled water with 0.1% Tween-80 was added. The mixture was shaken for 10 minutes on an electric shaker to release spores and filtered through a sterile muslin cloth. A 10 \u0026micro;L aliquot of the suspension was transferred to a hemocytometer, and spores were counted in five squares per chamber. Dilutions were performed using C₁V₁ = C₂V₂ formula to adjust concentrations to 10⁵ and 10⁶ spores/mL.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePathogen Inoculation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTwo inoculation methods were tested: stem injection and soil drenching. The stem injection method followed Li et al. (2021), where a sterile disposable needle created a hole, and 1 \u0026micro;L of \u003cem\u003eF. stilboides\u003c/em\u003e spore solution was injected. For the soil drenching method, roots were slightly injured before applying 10 mL of the spore solution to the soil surface. Control seedlings received sterile distilled water. The seedlings were maintained at room temperature for two weeks before being moved to a net house, arranged in a complete randomized design, and managed under standard agronomic practices.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePathogen Confirmation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo confirm infection, \u003cem\u003eF. stilboides\u003c/em\u003e was re-isolated from symptomatic seedlings. Seedlings were uprooted, washed, and longitudinally sectioned at the inoculation site. Tissue samples were surface-sterilized (2% sodium hypochlorite for 1 min, 70% ethanol for 1 min, and rinsed four times in sterile distilled water) and blot-dried. Five pieces of tissue sample per treatment were plated on PDA, incubated at 24\u0026deg;C, and monitored for \u003cem\u003eF. stilboides\u003c/em\u003e growth.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicroscopic Examination of\u003c/b\u003e \u003cb\u003eFusarium\u003c/b\u003e \u003cb\u003eIsolates Spore\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSpore concentration was determined using a Neubauer hemocytometer under a light microscope (\u0026times;40 magnification). A 10 \u0026micro;L aliquot of the spore suspension was loaded into the hemocytometer chamber. Spores were counted in five squares, and the spore concentration (spores/mL) was calculated.To assess the morphology of \u003cem\u003eFusarium\u003c/em\u003e spores, 10 \u0026micro;L of the spore suspension was placed on a clean glass slide, stained with lactophenol cotton blue, and covered with a sterile cover slip. Observations were conducted under a light microscope. Spore characteristics including shape and size were documented.\u003c/p\u003e\u003cp\u003e\u003cb\u003eVoucher Specimen and Identification\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePlant material was collected in four coffee-growing regions in Tanzania: Suji (Same District- Kilimanjaro region), Mahande and Mbangamao (Mbinga DC- Ruvuma region), Ayalabe (Karatu District- Arusha), and Bara (Mbozi District- Songwe region). Neyonkulu Sedikia Kahishai, Nuhu Aman Mbwebwe, Deusdedit Kilambo, and Fatuma Jumapili Ramadhani identified the specimen, and vouchers were deposited at the Tanzania Coffee Research Institute Herbarium.\u003c/p\u003e\u003cp\u003e\u003cb\u003eData Collection and Analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMorphocultural data were recorded based on colony morphology, growth rate, pigmentation, and sporulation. Spore density was quantified using a hemocytometer (Leslie and Summerell, 2006). Disease severity was assessed using a modified 0\u0026ndash;3 scale: 0\u0026thinsp;=\u0026thinsp;Symptomless plants, 1\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;10% limited wilting, 2\u0026thinsp;=\u0026thinsp;10\u0026ndash;30% severe yellowing and wilting, 3\u0026thinsp;=\u0026thinsp;30\u0026ndash;50% very severe yellowing and wilting. Disease incidence was calculated using the formula by Cooke et al. (2006). Data were subjected to ANOVA in GENSTAT 2014 16th edition software to generate means, variances and standard error of difference. The means were separated using Tukey\u0026rsquo;s Honest Significant Difference test at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results and discussions","content":"\u003cp\u003e\u003cb\u003eSporulation and Conidial Morphology of\u003c/b\u003e \u003cb\u003eFusarium stilboides\u003c/b\u003e \u003cb\u003eIsolates\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe five \u003cem\u003eFusarium stilboides\u003c/em\u003e isolates exhibited notable variation in sporulation capacity and conidial morphology. Among the isolates, KSS24 and SVB24 demonstrated abundant sporulation, whereas AKA24 exhibited moderate sporulation. In contrast, RMMb24 and RMM24 showed sparse sporulation under the given conditions (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMicroscopic examination revealed that four isolates—KSS24, SVB24, AKA24, and RMMb24 produced round microconidia, which are small, single-celled, and non-septate structures typical of certain \u003cem\u003eFusarium species\u003c/em\u003e during specific developmental stages (Leslie and Summerell, 2006). The presence of microconidia in these isolates suggests an adaptation to environmental or physiological conditions favoring their production. On the other hand, the RMM24 isolate, despite its sparse sporulation, produced canoe-shaped, septate macroconidia, a characteristic feature of many \u003cem\u003eFusarium species\u003c/em\u003e. High-magnification microscopy confirmed the distinct elongated shape and segmentation of these macroconidia.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSporulation Levels and Conidial Morphology of \u003cem\u003eFusarium stilboides\u003c/em\u003e Isolates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsolate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSporulation Level (spore/ml)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eConidial Shape\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRMM24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSparse (3.0x10\u003csup\u003e6\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCanoe-shaped macroconidia\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKSS24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbundant(3.6x10\u003csup\u003e6\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRound microconidia\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAKA24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eModerate (2.26x10\u003csup\u003e7\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRound microconidia\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSVB24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAbundant (5.6x10\u003csup\u003e7\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRound microconidia\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRMMb24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eModerate (1.08x10\u003csup\u003e7\u003c/sup\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRound microconidia\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe observed variation in sporulation among \u003cem\u003eFusarium stilboides\u003c/em\u003e isolates aligns with previous studies highlighting morphocultural diversity within \u003cem\u003eFusarium species\u003c/em\u003e. (Leslie and Summerell, 2006; Aoki \u003cem\u003eet al.\u003c/em\u003e, 2014; Lazarotto \u003cem\u003eet al.\u003c/em\u003e, 2014). The high sporulation levels observed in KSS24 and SVB24 suggest a potential advantage in dissemination and infection, as conidial density is a critical factor in pathogenicity (Cai \u003cem\u003eet al.\u003c/em\u003e, 2022). The tendency of the RMM24 isolate to produce macroconidia instead of microconidia under laboratory conditions may indicate the influence of specific environmental or physiological factors on sporulation, a pattern also reported in other \u003cem\u003eFusarium species\u003c/em\u003e (Ortoneda \u003cem\u003eet al.\u003c/em\u003e, 2004; Tshilenge \u003cem\u003eet al.\u003c/em\u003e, 2010).\u003c/p\u003e\u003cp\u003eVariations in sporulation patterns and conidial morphology can significantly influence the pathogenicity and epidemiology of \u003cem\u003eFusarium stilboides\u003c/em\u003e. Isolates producing higher quantities of microconidia, such as KSS24 and SVB24, may exhibit enhanced potential for rapid disease spread, as microconidia are often more easily dispersed (Summerell, 2019; Serani \u003cem\u003eet al.\u003c/em\u003e, 2007). In many \u003cem\u003eFusarium species\u003c/em\u003e, microconidia serve as primary inoculum, facilitating both initial infection and secondary spread under favorable environmental conditions.\u003c/p\u003e\u003cp\u003eThe RMM24 isolate, which produces canoe-shaped macroconidia, may have a lower potential for immediate infection and rapid disease spread. Macroconidia, being generally larger, often require specific conditions for germination, potentially resulting in a slower infection process compared to microconidia. However, their presence could indicate a more aggressive or persistent infection strategy, as macroconidia frequently function as survival structures, enabling the pathogen to endure unfavorable environmental conditions (Ploetz, 2016).\u003c/p\u003e\u003cp\u003eDifferences in sporulation among isolates may also reflect underlying genetic variability associated with geographic origin. Studies on \u003cem\u003eFusarium species\u003c/em\u003e infecting coffee, such as \u003cem\u003eF. xylarioides and F. stilboides\u003c/em\u003e, have demonstrated that environmental factors including temperature, humidity, and media composition can influence sporulation rates and conidial morphology (Acuña \u003cem\u003eet al.\u003c/em\u003e, 2021; Gathuru \u003cem\u003eet al.\u003c/em\u003e, 2021). The findings of this study suggest similar variability among Tanzanian \u003cem\u003eF. stilboides\u003c/em\u003e isolates, with potential implications for disease epidemiology and management strategies.\u003c/p\u003e\u003cp\u003e\u003cb\u003eGrowth Rate and Morphological Characteristics of\u003c/b\u003e \u003cb\u003eFusarium stilboides\u003c/b\u003e \u003cb\u003eIsolates\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe growth rate of \u003cem\u003eFusarium stilboides\u003c/em\u003e isolates over seven days, illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, further emphasizes their variability. SVB24 exhibited the highest growth rate, reaching the greatest colony diameter by Day 7, followed by RMMb24, RMM24 and KSS24, while AKA24 displayed the slowest expansion. There was a significant difference in growth rate (P \u0026lt; 0.05) among the isolates over time.These differences in growth rates may be linked to sporulation capacity and conidial morphology suggesting that isolates with abundant microconidia such as SVB24 have enhanced colonization potential.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eMorphological Study of\u003c/b\u003e \u003cb\u003eF. stilboides\u003c/b\u003e \u003cb\u003eIsolates\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMorphological characteristics of \u003cem\u003eF. stilboides\u003c/em\u003e isolates (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) indicate notable variation in colony color, texture, and pigmentation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Isolates with higher growth rates, such as SVB24 and RMMb24, were predominantly cottony or slimy in texture with white or pink color. In contrast, the slow-growing isolate AKA24 exhibited a cream-colored cottony colony, potentially indicative of lower adaptability to laboratory conditions.\u003c/p\u003e\u003cp\u003eDifferences in sporulation, growth rates, and colony morphology highlight the adaptive strategies of \u003cem\u003eF.stilboides\u003c/em\u003e isolates under varying environmental conditions. Previous studies on \u003cem\u003eFusarium species\u003c/em\u003e have demonstrated that pigmentation, texture, and edge type may be linked to pathogenic fitness, resistance to environmental stress, and virulence (Acuña \u003cem\u003eet al.\u003c/em\u003e, 2021). Thus, these findings emphasize the need for further studies to explore the genetic and environmental factors influencing these traits.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cem\u003eFusarium Stilboides\u003c/em\u003e cultures Morphological Characteristics\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsolate\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eColony color\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003epigmentation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eColony texture\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEdge type\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKSS24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePink\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCottony\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSmooth\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRMM24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBlack\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePurple\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVelvety\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWavy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRMMb24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePink\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePink\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSlimy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSmooth\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSVB24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWhite\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePink\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCottony\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSmooth\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAKA24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCream\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePink\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCottony\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eWavy\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cbr\u003e\u003cp\u003e\u003cb\u003eVirulence of\u003c/b\u003e \u003cb\u003eF. stilboides\u003c/b\u003e \u003cb\u003eIsolates\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe severity of \u003cem\u003eF. stilboides\u003c/em\u003e isolates varied significantly across the different treatments over period (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The distilled water control exhibited no symptoms throughout the experimental period. On the other hand, the isolate KSS24 exhibited the highest disease severity progression, reaching a mean score of 3.00 from 63 DAI onwards.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDisease Severity Progression of \u003cem\u003eFusarium Stilboides Isolates\u003c/em\u003e over Time\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"14\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIsolate code\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e28DAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35DAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e42DAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e49DAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e56DAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e63DAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e70DAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e77DAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e84DAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003e91DAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c12\"\u003e\u003cp\u003e98DAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c13\"\u003e\u003cp\u003e105DAI\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c14\"\u003e\u003cp\u003e112DAI\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAKA24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDistilled water\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e0.00 a\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRMM24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e2.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e2.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e2.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e2.00 ab\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRMMb24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.33 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.33 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.33 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.33 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.33 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.33 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.33 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.33 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.33 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.33 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.33 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e1.33 ab\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSVB24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e1.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e1.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e1.00 ab\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e1.00 ab\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKSS24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1.67 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3.00 a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e3.00 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3.00 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e3.00 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e3.00 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e3.00 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e3.00 b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e3.00 b\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eGrand mean\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.61\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.94\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e1.17\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1.17\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e1.17\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e1.33\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e1.33\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e1.33\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e1.33\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cb\u003e1.33\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e1.33\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u003cb\u003e1.33\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e\u003cb\u003e1.33\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eS.e.d\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.609\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.745\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.72\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.72\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.72\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.638\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e0.638\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0.638\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e0.638\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cb\u003e0.638\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e0.638\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u003cb\u003e0.638\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e\u003cb\u003e0.638\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ecv%\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e122\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e96.7\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e75.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e75.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e75.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e58.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e58.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e58.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e58.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cb\u003e58.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e58.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u003cb\u003e58.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e\u003cb\u003e58.6\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eF prob.\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e0.148\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e0.372\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e0.104\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e0.104\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e0.104\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c12\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c13\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c14\"\u003e\u003cp\u003e\u003cb\u003e0.008\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"14\"\u003e\u003cb\u003e*Means with the same letter in the same column indicate no significant differences (p ≤ 0.05) according to Tukey’s Honestly Significance Difference. *DAI = Days After Inoculation; cv%=coefficient of variation; F prob = F probability\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong the test isolates, RMM24 showed a gradual increase in disease severity, attaining a mean score of 2.00 from 35 days after inoculation and no further disease development. Isolates RMMb24 and SVB24 exhibited relatively lower disease severity, with both reaching a maximum of 1.33 and 1.00 mean score, respectively at 112 days after inoculation. AKA24 showed the lowest disease severity among the isolates, remaining at a mean score of 0.67 throughout the experiment, comparable to the distilled water control.\u003c/p\u003e\u003cp\u003eThere were significant differences (P \u0026lt; 0.05) among the isolates over time. The F probability values indicated no significant differences among treatments at early evaluation stages, but significant variations emerged from 63 days after inoculation onward (p \u0026lt; 0.05). The coefficient of variation (CV%) decreased over time, indicating more stability in disease severity scores as the experiment progressed. The high severity recorded in KSS24 compared to other isolates indicates its higher aggressiveness, aligning with previous studies on \u003cem\u003eFusarium species\u003c/em\u003e (Ortoneda \u003cem\u003eet al.\u003c/em\u003e, 2004), where pathogenic variability has been reported among isolates from different geographical regions. The relatively lower severity in RMMb24 and SVB24 suggests that these isolates may be less virulent or slower in colonization under the given experimental conditions.\u003c/p\u003e"},{"header":"Conclusion and Recommendations","content":"\u003cp\u003eThis study highlights significant variations in the sporulation, conidial morphology, growth rate, and virulence of \u003cem\u003eFusarium stilboides\u003c/em\u003e isolates collected from different coffee grown regions of Tanzania. The observed differences suggest that certain isolates, such as KSS24, possess enhanced sporulation and pathogenic potential, which may contribute to increased disease spread in coffee plants. On the other hand, isolates like RMM24 and AKA24 exhibited lower sporulation and disease severity, indicating potential variability in aggressiveness and adaptability. The presence of both microconidia and macroconidia across different isolates suggests diverse survival and infection strategies, which may influence disease epidemiology and management approaches.\u003c/p\u003e\u003cp\u003eUnderstanding the variability within \u003cem\u003eFusarium stilboides\u003c/em\u003e populations will be crucial in mitigating the impact of this pathogen on coffee production in Tanzania and beyond. The findings of this study show the importance of characterizing Fusarium isolates at both morphological and pathogenic levels to develop targeted disease management strategies. Future research should focus on molecular characterization and environmental interactions influencing pathogenicity, which could further inform breeding programs aimed at developing resistant coffee varieties.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch3\u003eFUNDING AND CONSENT DECLARATION\u003c/h3\u003e\n\u003cp\u003eTanzania Coffee Research Institute provided funding.\u003c/p\u003e\n\u003cp\u003eEthics, Consent to Participate, and Consent to Publish declarations: not applicable.\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003eData availability statement: The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003eVoucher specimen and identification: Plant material was collected in four coffee-growing regions in Tanzania: Suji (Same District- Kilimanjaro region), Mahande and Mbangamao (Mbinga DC- Ruvuma region), Ayalabe (Karatu District- Arusha), and Bara (Mbozi District- Songwe region). Neyonkulu Sedikia Kahishai, Nuhu Aman Mbwebwe, Deusdedit Kilambo, and Fatuma Jumapili Ramadhani identified the specimen, and vouchers were deposited at the Tanzania Coffee Research Institute Herbarium.\u003c/p\u003e\n\u003cp\u003eEthics and Guidelines: No human participants or live vertebrates were involved in this study. Collection of fungal isolates followed institutional (TaCRI) and national (Tanzania) guidelines. Plant specimens were identified and voucher specimens deposited under institutional (TaCRI) policies in compliance with Tanzanian biosafety protocols.\u003c/p\u003e\n\u003ch3\u003eAuthor Contribution\u003c/h3\u003e\n\u003cp\u003eN.S.K and D.K wrote the main manuscript, N.A.M and F.J.R prepared some figures and all authors reviewed the manuscript.\u003c/p\u003e\n\u003ch3\u003eData Availability\u003c/h3\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eAcu\u0026ntilde;a R, Mena J, Gil L. \u003cem\u003eFusarium\u003c/em\u003e species infecting coffee crops: Morphological and molecular characterization. J Plant Pathol. 2021;103(2):345\u0026ndash;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s42161-021-00915-3.\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAcu\u0026ntilde;a R, Smith DR, Jones TK. Environmental factors influencing \u003cem\u003eFusarium\u003c/em\u003e sporulation and pathogenicity in coffee crops. Plant Pathol J. 2021;37(2):215\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAoki T, O\u0026rsquo;Donnell K, Homma Y, Lattanzi AR. Morphological and molecular characterization of \u003cem\u003eFusarium\u003c/em\u003e species associated with coffee plants. Mycol Res. 2014;118(1):1\u0026ndash;11. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.mycres.2014.01.004\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAoki T, O\u0026rsquo;Donnell K, Geiser DM. Systematics of key \u003cem\u003eFusarium\u003c/em\u003e pathogens of coffee. Mycol Res. 2014;118(9):865\u0026ndash;78.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003e\u0026bull;AL-Faifi Z, Alsolami W, Abada E, Khemira H, Almalki G, Modafer Y. (2022). Fusarium oxysporum and Colletotrichum musae associated with wilt disease of Coffea arabica in coffee gardens in Saudi Arabia. International Journal of Agronomy, 2022, 3050495. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2022/3050495\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003e\u0026bull;Bertrand B, Nunez C, Sarah J-L. Disease complex in coffee involving \u003cem\u003eMeloidogyne arabicida\u003c/em\u003e and \u003cem\u003eFusarium oxysporum\u003c/em\u003e. Plant Pathol. 2000;49(3):383\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-3059.2000.00456.x\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCai F, Gao R, Zhao Z, Ding M, Jiang S, Zhao L. Conidial density and pathogenic potential of \u003cem\u003eFusarium\u003c/em\u003e species. Fungal Biology. 2022;126(4):324\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCai X, Wang Y, Li H, Zhang Y. Conidial density and pathogenicity in \u003cem\u003eFusarium\u003c/em\u003e species: A comparative study. J Plant Pathol. 2022;104(2):455\u0026ndash;68.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCooke BM, Jones DG, Kaye B. Diseases of Field Crops. CRC; 2006.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGathuru EM, Mwangi LM, Otieno N. Pathogenic variability in \u003cem\u003eFusarium\u003c/em\u003e spp. causing coffee diseases. Afr J Plant Sci. 2021;15(3):87\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGathuru EM, Omondi CO, Nyabundi JO. Occurrence and management of \u003cem\u003eFusarium\u003c/em\u003e bark disease in Kenya. J Coffee Res. 2021;50(2):89\u0026ndash;102.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLazarotto M, Milanesi PM, Muniz MFB, Reiniger LRS, Beltrame R, Harakava R, Blume E. Morphological and molecular characterization of \u003cem\u003eFusarium\u003c/em\u003e spp. pathogenic to pecan tree in Brazil. Genet Mol Res. 2014;13(4):9390\u0026ndash;402. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4238/2014.November.18.13\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLeslie JF, Summerell BA. The Fusarium Laboratory Manual. Blackwell Publishing; 2006.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLi X, Zhang Y, Wang J, Chen H. Development of an inoculation technique for rapidly evaluating maize inbred lines for resistance to \u003cem\u003eFusarium\u003c/em\u003e spp. Plant Dis. 2021;105(12):3683\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PDIS-09-20-2016-SR\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003e\u0026bull;Oliveira L, de Rodrigues JMG, Silva AAC, Oliveira EKCe, de Barros ACS, Fraga MC, de Nascimento E O., and, Silva MRM. (2021). Morphological and phylogenetic characterization of \u003cem\u003eFusarium Link\u003c/em\u003e. \u003cem\u003eAustralian Journal of Crop Science, 15\u003c/em\u003e(12), Article p3292. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21475/ajcs.21.15.12.p3292\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eOrtoneda M, Guarro J, Madrid MP, Caracuel Z, Roncero MIG, Mayayo E, Di Pietro A. \u003cem\u003eFusarium\u003c/em\u003e pathogenicity and sporulation under different environmental conditions. Appl Environ Microbiol. 2004;70(5):3206\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePloetz RC. \u003cem\u003eFusarium\u003c/em\u003e species associated with coffee diseases: Taxonomy and control. Phytopathology. 2016;106(2):171\u0026ndash;87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PHYTO-09-15-0230-RVW\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSerani S, Taligoola HK, Hakiza GJ. An investigation into \u003cem\u003eFusarium\u003c/em\u003e spp. associated with coffee and banana plants as potential pathogens of robusta coffee. Afr J Ecol. 2007;45(1):91\u0026ndash;5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-2028.2007.00865.x\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSiddiqi MA, Corbett DCM. Coffee bark diseases in Malawi: Properties of the causal organism and conditions favoring the disease. Trans Br Mycological Soc. 1968;51(1):129\u0026ndash;35. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0007-1536(68)80017-8\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSummerell BA. \u003cem\u003eFusarium\u003c/em\u003e: Identification and diversity in agricultural and natural ecosystems. Phytopathology. 2019;109(4):512\u0026ndash;26. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1094/PHYTO-01-19-0030-R\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSummerell BA. Fifty years of \u003cem\u003eFusarium\u003c/em\u003e: How understanding the ecology of \u003cem\u003eFusarium species\u003c/em\u003e has informed plant pathology. Fungal Biology Reviews. 2019;33(1):20\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTshilenge L, Kalonji A, Tshilenge PJ. Determination of cultural and biometrical characters of \u003cem\u003eFusarium\u003c/em\u003e species isolated from plant material harvested from coffee (\u003cem\u003eCoffea canephora\u003c/em\u003e Pierre) infected with coffee wilt disease in the Democratic Republic of Congo. Afr J Agric Res. 2010;5(22):3145\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWaller JM, Bigger M, Hillocks RJ. Coffee Pests, Diseases, and Their Management. CABI Publishing; 2007.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYang J, Han J, Jing Y, Li S, Lan B, Zhang Q, Yin K. Virulent \u003cem\u003eFusarium\u003c/em\u003e isolates with diverse morphologies show similar invasion and colonization strategies in alfalfa. Front Plant Sci. 2024;15:1390069. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2024.1390069\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fusarium Bark Disease, Fusarium stilboides, Coffea arabica, Morphocultural characterization","lastPublishedDoi":"10.21203/rs.3.rs-6807950/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6807950/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eFusarium stilboides\u003c/em\u003e, the causative agent of Fusarium bark disease (FBD) in \u003cem\u003eCoffea arabica\u003c/em\u003e, poses a significant threat to coffee production. Despite its impact, limited research has explored the pathogen\u0026rsquo;s morphological diversity and pathogenic variability in Tanzania. This study aimed to characterize the morphocultural traits and aggressiveness of \u003cem\u003eF. stilboides\u003c/em\u003e isolates collected from four coffee-growing regions in Tanzania: Suji (Same District- Kilimanjaro region), Mahande and Mbangamao (Mbinga DC- Ruvuma region), Ayalabe (Karatu District- Arusha), and Bara (Mbozi District- Songwe region). Five isolates were obtained from symptomatic coffee trees. They were then cultured on Potato Dextrose Agar (PDA), and assessed for colony morphology, pigmentation, growth rate, and sporulation. Evaluation of pathogenicity was through stem injection and soil-drenching methods of inoculation on \u003cem\u003eC. arabica\u003c/em\u003e seedlings, with disease severity monitored over 112 days. Results revealed significant variations in colony color, texture, and growth rate among isolates. Sporulation capacity also differed significantly, with isolates KSS24 and SVB24 exhibiting abundant sporulation, while RMM24 had the lowest. Pathogenicity assays showed a significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Isolate KSS24 was the most aggressive, reaching a mean disease severity score of 3.00 by 63 days after inoculation, whereas AKA24 exhibited the lowest severity (0.67). These findings highlight the morphological and pathogenic variability of \u003cem\u003eF. stilboides\u003c/em\u003e in Tanzania, underscoring the need for further studies on molecular characterization and environmental interactions influencing pathogenicity, which could further inform breeding programs aimed at developing resistant coffee varieties.\u003c/p\u003e","manuscriptTitle":"Morphocultural Characterization and Aggressiveness of Fusarium stilboides Isolates from Four Coffee Growing Regions in Tanzania","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 13:40:25","doi":"10.21203/rs.3.rs-6807950/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"41307849-b7fb-46cc-b637-18919415aa2d","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T10:24:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-22 13:40:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6807950","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6807950","identity":"rs-6807950","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}