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Impact of temperature on vegetative and pathogenic development of the fungal multi-host pathogen Lasiodiplodia theobromae (Pat.) Griffon & Maubl.A
EdgarRodríguez-Gálvez1✉CesarL.Haro-Díaz2
DenisseTuesta-Berrú1
HolgerB.Deising3✉Emailholger.deising@landw.uni-halle.de
ERG1Emailerg@unp.edu.pe
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Department of Plant Health, Faculty of AgronomyNational University of PiuraPeru2Department of Statistics, Faculty of SciencesNational University of PiuraPeru
3Faculty of Natural Sciences III, Institute of Agricultural and Nutritional Sciences, Chair of Phytopathology and Plant ProtectionMartin Luther University Halle-WittenbergHalle (Saale)Germany
Edgar Rodríguez-Gálvez1*, Cesar L. Haro-Díaz2, Denisse Tuesta-Berrú1 and Holger B. Deising3*
1Department of Plant Health, Faculty of Agronomy, National University of Piura, Peru
2Department of Statistics, Faculty of Sciences, National University of Piura, Peru
3Martin Luther University Halle-Wittenberg, Faculty of Natural Sciences III, Institute of Agricultural and Nutritional Sciences, Chair of Phytopathology and Plant Protection, Halle (Saale), Germany
*Corresponding authors: erg@unp.edu.pe (ERG); holger.deising@landw.uni-halle.de (HBD)
Abstract
Lasiodiplodia theobromae is an economically important multi-host plant pathogen infecting nearly 500 hosts, including perennial crops. In Peru, L. theobromae has been identified as the cause of dieback in blueberries, avocados, mangoes, and table grapes. These crops are grown in the northern coastal regions of Peru at temperatures expected to increase due to global warming. We used the aggressive isolate LA-SOL5 of L. theobromae originally isolated from grapevine to study temperature dependence of in vitro vegetative growth, conidiation, conidial germination and virulence on grapevine cultivar Red Globe. Mycelial growth and formation of pycnidia were optimal at 30°C, but pre-infection rates of conidiation and conidial germination increased up to 35°C and correlated well with virulence on grapevine. Our studies show that simple conidiation and conidial germination assays could thus be used to predict development of disease severity and suggest increased severity of L. theobromae-associated dieback in grapes at future climate changes.
Keywords:
Lasiodiplodia theobromae
dieback
sporulation
temperature
virulence
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On the global scale, plant diseases pose a severe threat to modern agriculture, and adaptation of disease control measures to environmental conditions requires precise knowledge of infection biology. Environmental factors such as the surface air temperature clearly shape incidences of diseases in both plants and humans (Chaloner et al, 2020; Raza and Bebber 2022; Velasquez et al. 2018; Seidel et al. 2024). Plausibly, this scenario may be of particular importance in pathogens causing disease in multiple hosts such as the grey mold fungus Botrytis cinerea (Pers.) in moderate climates or Lasiodiplodia theobromae (Pat.) Griffon and Maubl. causing dieback disease in woody plants in the tropics (Williamson et al. 2007; Punithalingam 1976). Interestingly, a recent study on temperature-dependent infection risk imposed by 80 fungal and oomycete pathogens in 12 crops over the twenty-first century revealed that infection risks are likely to increase at high latitudes whereas in the tropics this risk will remain unchanged or decline (Chaloner et al. 2021). Furthermore, altitudes determine local temperatures and thus temperature-dependent infection risks (Chaloner et al. 2021). Another study showed that the optimum temperature for vegetative growth in culture correlates only moderately with disease development (correlation coefficients of 0.65), but the optimum temperatures for sporulation and spore germination both show correlation coefficients of 0.72 (Chaloner et al. 2020). Thus, data on temperature-dependence of sporulation and spore germination of economically relevant pathogens like L. theobromae may help predicting future disease development at climate change-dependent temperature elevation.
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The fungus L. theobromae, a member of the family Botryosphaeriaceae (Zhang et al. 2021), infects approx. 500 host plants (Punithalingam 1976). Indeed, the enormous range of hosts, including high-profit crops such as blueberries (Rodríguez-Gálvez et al. 2020; Liddle et al. 2019), mangos (Rodríguez-Gálvez et al. 2017), avocados (Rodríguez-Gálvez et al. 2021), and, importantly, table grapes (Rodríguez-Gálvez et al. 2015), is surprising because this pathogen requires wounds for host invasion (Burruano et al. 2008). The fungus is distributed worldwide, but is most predominant in tropical and subtropical regions (Rathnayaka et al. 2023; Aroca et al. 2008; Pitt et al. 2010; Taylor et al. 2005; Úrbez-Torres et al. 2008; Úrbez-Torres et al. 2006). Disease symptoms, depending on the host, include damping off, blight, dieback, root rot, collar rot, stem necrosis, gummosis, stump rot, leaf spot, fruit blight, and fruit rot (Punithalingam 1976). In grapevine cultivation, L. theobromae it is considered the most aggressive species of the Botryosphaeeriaceae family (Úrbez-Torres 2011).As disease symptom severity in most fungus-crop interactions is temperature-dependent (Chaloner et al. 2021), we depict average mean surface air temperature data of Perú, starting from 1950 (climataknowledgeportal.worldbank.org/country/peru/climate-data-historical). In spite of some variability across the years, data indicate a clear mean surface air temperature increase (Fig. 1).
Fig. 1
Development of the average mean surface air temperature in Perú since 1950. Data are from climataknowledgeportal.worldbank.org.
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In order to investigate whether increases in temperature affect vegetative mycelial growth and sporulation rates, we cultivated isolate LA-SOL5 of L. theobromae on 2% (w/v) Potato Dextrose Agar (PDA) (HiMedia Laboratories Pvt. Ltd., Dindhori, Nashik, India) as described (Rodríguez-Gálvez et al. 2025). This isolate had been obtained from grapevine branches, caused early dieback symptoms and showed the highest virulence in comparative pathogenicity tests employing 70 isolates (Rodríguez-Gálvez et al. 2015). Colonies were grown on PDA at 30°C, and 5-mm-diameter agar discs were excised from the edge of a 72-hour-old colony and placed at the center of 90-mm Petri dishes containing PDA. The plates were sealed and incubated in the dark at 20°C, 25°C, 30°C and 35°C (Velp Scientifica FOC 120i, Italy). Five plates per temperature level were used and the experiment was repeated four times. Colony diameters were measured daily until the mycelium had covered the Petri dish.For comparison of means and analyses of statistical significance, the STATGRAPHICS Centurion XVI program (Statgraphics Technologies, Inc., The Plains, VA, USA) was used. Duncan analyses were performed at a P value of 0.05.
On PDA, mycelial growth rates of L. theobromae showed a maximum at 30°C and declined at increasing the temperature to 35°C. Remarkably, at 30°C the fungus grew more than twice as fast as at 20°C (Fig. 2).
Fig. 2
Vegetative mycelial growth of L. theobromae at 20, 25, 30, and 35°C. A. Phenotype of colonies. Photos were taken at 2 days post inoculation. B. Quantification of radial growth rates. Error bars are standard deviations, different letters indicate statistical difference at P = 0.05.
Supporting these data, a optimum mycelial growth temperature of 30°C was also reported for L. theobromae isolates isolated from banana and guava plants in India, Pakistan, Bangladesh or Australia, respectively (Alam et al. 2001; Baloch et al. 2018; Renganathan et al. 2020; Vijay et al. 2021; Pitt et al. 2010). Recent metastudies suggested that across the 16 species belonging to the Botryosphaeriaceae family, L. theobromae, and Botryosphaeria dothidea exhibited the highest temperature requirements, i.e., temperatures of more than 30°C (Ji et al. 2023). These tropical isolates can thus be regarded as well-adapted to high environmental temperatures.
Sporulation and spore germination rates correlated well with virulence in several plant pathogenic fungi (Chaloner et al. 2020). In L. theobromae, strongly pigmented, uni-septate conidia are formed in fruiting bodies called pycnidia. Therefore, in order to characterize temperature-dependence of asexual sporulation of L. theobromae at different temperatures, we first quantified formation of pycnidia. L. theobromae was inoculated onto Petri dishes as described above and incubated in a bioclimatic chamber (Binder KBF P 240, Class 3.1- Germany) at 20°C, 25°C, 30°C, and 35°C at constant light to induce pycnidia formation (Alam et al. 2001). After seven days, the number of pycnidia per cm² was determined, using a stereomicroscope (Zeiss model, Stemi 305, Obercochen Germany). The tests were repeat three times.
L. thebromae produced the greatest number of pycnidia at 30°C (72.71 ± 4.23 pycnidia/cm2). Interestingly, increasing the temperature by only 5°C, i.e., from 25°C to 30°C caused more than doubling of pycnidium differentiation and indicated the enormous temperature dependence of formation of fruiting bodies (Figs. 3A and B).
To analyze temperature dependence of conidial germination, aqueous spore suspensions were incubated in PDA at the four temperatures indicated above for 24 hours, and germination rates were determined using a compound optical microscope (Olympus model BX53F2, Tokyo, Japan). Intriguingly, at 6 hpi approx. 30% of the conidia incubated at 35°C had germinated, whereas at 20, 25 and 30°C germination rates were below 10%, suggesting thermophilicity of the early infection stages of this fungus. Throughout the entire time span of the experiment, i.e., up to 24 hpi, germination rates were highest at 35°C, exceeding 80% (Figs. 3C and D). For comparison, at 20°C only one-third of the conidia had germinated at 24 hpi (Fig. 3E). Supporting our data, Gubler and co-workers reported that 40°C was the optimal germination temperature of conidia of a L. theobromae isolate from grapevine cankers (Úrbez -Torres et al. 2011).
Fig. 3
Temperature dependence of formation of pycnidia and of conidial germination of L. theobromae. A. Pycnidia formed on the surface of PDA at different temperatures. Size bar is 1 cm. B. Quantification of pycnidia formed at different temperatures per cm2. C. Micrographs of germination of strongly pigmented conidia at different temperatures. Arrowheads point at germinated conidia. Size bar is 5 µm. D. Quantification of conidial germination. Conidial germination rates were determined every three hours, and tests were repeated five times. Error bars in B and D are standard deviations; distinct letters indicate statistical differences at P = 0.05.
In order to investigate the effect of increasing temperature on disease symptom severity, four-month-old Red Globe grapevine plants, grafted onto Salt Creek rootstock (Viveros Génesis, Cayaltí, Lambayeque, Perú), with a shoot height of 30 cm and stem diameter of approximately 5 mm, were used in infection experiments. Prior to inoculation, plants were acclimated to the temperature used for infection assays in a growth chamber (Binder KBF P 240, CLASS 3.1, Germany) at a 10 hours light / 14 hours darkness regime and 65% relative humidity for five days. For apex inoculation, the apical bud was excised, a 2 mm PDA disc covered by mycelium of isolate LA-SOL5 was taken from the edge of a 48-hour old colony and placed onto the wound as described (Rodríguez-Gálvez et al. 2025). Inoculated plants were further incubated at 20°C, 25°C, 30°C or 35°C. Eight inoculated plants were used per temperature, the experiment was repeated three times. Basipetal expansions of internal necroses were quantified at 14 days post inoculation as described (Rodríguez-Gálvez et al. 2025) and served as a measure of infection severity.
Fig. 4
Disease severity caused by L. theobromae at different temperatures. A. Internal necroses of apex-inoculated grapevine plants. Arrows indicate progression of internal necrosis, arrowheads indicate intact (30°C) and necrotic (35°C) leaves. B. Length of internal necrotic lesions measured at 14 dpi. Error bars indicate standard deviations; distinct letters indicate statistical differences (P = 0.05).
At all temperatures tested, L. theobromae invaded the grapevine plant after apex inoculation. Expansion of dark internal lesions increased with temperature, and the first apical node was reached at 14 dpi at 25°C. At 30°C, the pathogen had overgrown the top-node, but the leaf had not been infected and was green. At 35°C, the infection had further progressed basipetally, and the leaf at the top-node had turned necrotic (Fig. 4A). Quantification of the length of the internal necroses (Fig. 4B) confirmed macroscopically observed temperature dependence of disease progression. Our results are consistent with reported by others (Qiu et al. 2016; Úrbez-Torres 2011) and indicate that virulence of L. theobromae on grapevine cuttings was highest at 35°C.
In conclusion, our study showed that in contrast to mycelial growth and formation of pycnidia showed a temperature optimum at 30°C. However, rates of formation of conidia and conidial germination, both pre-requisites for spread of infective propagules and effective infection, increased up to 35°C, and, consistently, disease symptom expression was most severe at 35°C. Thus, global temperature increases are likely to cause increased spreading and severity of L. theobromae-induced dieback of grapevine plants.
Funding. This work was funded by the Central Research Office (OCIN) of the National University of Piura-Peru through the University Research Fund (FEDU).
Acknowledgment.
The authors thank Angela Peña Gonzales for her excellent technical assistance.
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Authors' contribution.
The conceptualization of this research was developed by E. Rodriguez-Galvez (principal investigator) and H.B. Deising. The experimental design and statistical analyses were developed by C. Haro-Diaz. The preparation of the virulent isolate LA-SOL5 and the vegetative in vitro growth assays were performed by D. Tuesta-Berrú. The maintenance, inoculation and symptom evaluation assays in plants were performed by E. Rodríguez-Gálvez and D. Tuesta-Berrú. Data analysis and manuscript writing were performed by E. Rodríguez-Gálvez and H.B. Deising. The article was reviewed by all authors.
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Data availability.
All data generated and analyzed are available upon request to the corresponding author.
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DeclarationsConflict of interest.
The authors declare no conflict of interest.
References
Alam SM, Most-Ferdousi B, Montaz AS, Rafiqul Islam M, Shah Alam M (2001) Effect of temperature, light and media on growth, sporulation, formation of pigments and pycnidia of Botryodiplodia theobromae Pat. Pakist J Biol Sci 4:1224–1227
Aroca A, Raposo R, Gramaje D, Armengol J, Martos S, Luque J (2008) First report of Lasiodiplodia theobromae associated with decline of grapevine rootstock mother plants in Spain. Plant Dis 92:832
Baloch A, Dad S, Ali R, Baloch RA, Hussain JG, Anwar M, Babar Bhatti ZU (2018) Effect of medium, temperature and pH, on in vitro growth of Botryodiplodia theobromae isolated from guava Pak. J Biotechnol 15:123–127
Burruano S, Mondello V, Conigliaro G, Alfonso A, Spagnolo A, Mugnai L (2008) Grapevine decline in Italy caused by Lasiodiplodia theobromae. Phytopathol Mediterr 47:132–136
Chaloner TM, Gurr SJ, Bebber DP (2021) Plant pathogen infection risk tracks global cropyields under climate change. Nat Clim Change 11:710–715
Chaloner TM, Gurr SJ, Bebber DP (2020) Geometry and evolution of the ecological niche in plant-associated microbes. Nat Commun 11:2955
Ji T, Salotti I, Altieri V, Li MyV, Rossi (2023) Temperature-Dependent Growth and Spore Germination of Fungi Causing Grapevine Trunk Diseases: Quantitative Analysis of Literature Data. Plant Dis 107 1:1386–1398
Liddle RL, Akinsanmi OA, Vj Galea (2019) Non-host specificity of Botryosphaeriaceae on macadamia and blueberry. Aust Plant Pathol 48:65–73
Pitt WM, Huang R, Steel CC, Savochia S (2010) Identification, distribution and current taxonomy of Botryosphaeriaceae species associated with grape vine decline in New South Wales and South Australia. Aust J Grape Wine Res 16:258–271
Punithalingam E (1976) Botryodiplody theobromae. CMI Descriptions of Fungi and Bacteria: 52, Sheet-519. Commonwealth Mycological Institute, Kew, Surrey
Qiu Y, Steel CC, Ash GJ, Savocchia S (2016) Effects of temperature and water stress on the virulence of Botryosphaeriaceae spp. causing dieback of grapevines and their predicted distribution using CLIMEX in Australia. Hortic Act 1115:26
Rathnayaka AR, Chethana KWT, Manawasinghe IS, Wijesinghe SN, de Silva NI, Tennakoon DS, Phillips AJL, Liu JK, Jones EBG, Wang Y, Hyde KD (2023) Lasiodiplodia: Generic revision by providing molecular markers, geographically distribution and haplotype diversity. Mycosphere 14:1254–1339
Raza MM, Bebber DP (2022) Climate change and plant pathogens. Curr Opin Microbiol 70:102233
Renganathan1 P, Karan R, Dhaarani S, Saravanan KR, Premkumar R (2020) Effect of different media, temperature, pH, carbon source and nitrogen source on mycelial growth of Lasiodiplodia theobromae causing crown rot of banana. Ann RSCB 24:1494–1506
Rodríguez- Gálvez E, Guerrero P, Barradas C, Crous PW, Alves A (2017) Phylogeny and pathogenicity of Lasiodiplodia species associated with dieback of mango in Peru. Fungal Biol 121:452–465
Rodríguez–Gálvez E, Haro–Diaz C, Maza–Aguirre S, Canahuire–Castillo F, Sullon–Saucedo J, Deising HB (2025) Lasiodiplodia theobromae disease symptom development in young avocado (Persea Americana L.) plants depends on the inoculation method. Trop Plant Pathol 50:18
Rodríguez-Gálvez E, Hilario S, Batista E, Lopes A, Alves A (2021) Lasiodiplodia species associated with dieback of avocado in the coastal area of Peru. Eur J Plant Pathol 161:219–232
Rodríguez-Gálvez E, Hilario S, Lopes A, Alves A (2020) Diversity and pathogenicity of Lasiodiplodia and Neopestalotiopsis species associated with stem blight and dieback of blueberry plants in Peru. Eur J Plant Pathol 157:89–102
Rodríguez-Gálvez E, Maldonado E, Alves A (2015) Identification and pathogenicity of Lasiodiplodia theobromae causing dieback of table grapes in Peru. Eur J Plant Pathol 141:477–489
Seidel D, Wurster S, Jenks JD, Sati H, Gangneux J-P, Egger M, Alastruey-Izquierdo A, Ford NP, Chowdhary A, Sprute R, Cornely O, Thompson GR III, Hoenigl M†, Kontoyiannis DP (2024) Impact of climate change and natural disasters on fungal infections. Lancet Microbe 5:e594–e605
Taylor A, Hardy GESJ, Wood P, Burguess TI (2005) Identification and pathogenicity of Botryosphaeria species associated with grapevine decline in Western Australia. Aust Plant Pathol 34:187–195
Úrbez-Torres JR, Leavitt GM, Guerrero JC, Guevara J, Gubler WD (2008) Identification and pathogenicity of Lasiodiplodia theobromae and Diplodia seriata, the causal agents of bot canker disease of grapevines in Mexico. Plant Dis 92:519–529
Úrbez-Torres JR, Leavitt GM, Voegel TM, Gubler WD (2006) Identification and distribution of Botryosphaeria species associated with grapevines cankers in California. Plant Dis 90:1490–1503
Úrbez-Torres JR (2011) The status of Botryosphaeriaceae species infecting grapevines. Phytopathol Mediterr 50:S5–S45
Velásquez AC, Danve C, Castroverde M, He SY (2018) Plant and pathogen warfare under changing climate conditions. Curr Biol 28:R619–R634
Vijay S, Renukadevi P, Mangammal P, Krishnamoorthy SV (2021) Effect of media, temperature and pH on the growth and pycnidial production of Lasiodiplodia theobromae (Pat.), causative of black root rot in mulberry. Pharm Innov J 10:340–343
Williamson B, Tudzynski B, Tudzynski P, Van Kan JAL (2007) Botrytis cinerea: the cause of grey mould disease. Mol Plant Pathol 8:561–580
Zhang W, Groenewald JZ, Lombard L, Schumacher RK, Phillips AJL, Crous PW (2021) Evaluating species in Botryosphaeriales. Pers Mol Phyl Evol Fungi 46:63–115