Impaginato 13 Adv. Hort. Sci., 2024 38(1): 13­24 DOI: 10.36253/ahsc­13913 Pumpkins (Cucurbita spp.) diversity and their associated microbiota R. Aydi Ben Abdallah (*), H. Chikh­Rouhou, H. Jabnoun­Khiareddine, M. Daami­Remadi Laboratory Research of Production and Protection for a Sustainable Horticulture, IRESA, University of Sousse, Regional Research Centre on Horticulture and Organic Agriculture, Chott Mariem, Tunisia. Key words: Breeding, Cucurbita spp., fruit yield, plant genotype, soil microbial community. Abstract: Root­associated microbiota play a key role in plant growth, resilience, and health. In this study, the microbial community structure in the rhizosphere of 12 pumpkins accessions belonging to three Cucurbita species i.e. C. pepo, C. maxima, and C. moschata, was monitored using the soil dilution plating tech­ nique on specific media. All accessions tested were also screened for their pro­ duction and yield parameters. Based on Principal Component Analysis (PCA), 4 accessions of C. maxima (namely C5, C23, C14.2 and C6.2) were characterized by the greatest average fruit weight and yield, the highest actinomycetes, bac­ terial, Trichoderma spp. and Aspergillus spp. communities, and the lowest total fungal population in their rhizosphere. Positive correlations were noted between fruit fresh weight, culturable bacteria and Trichoderma spp. popula­ tions in the rhizopshere of pumpkins accessions. Negative correlations were noted between fruit weight and yield parameters and the total culturable fun­ gal populations. The current study clearly demonstrated that the rhizosphere soil microbial communities have been shaped by Cucurbita species and acces­ sions. Based on the significant links observed between soil microbiota and yield parameters, future pumpkin breeding programs could be focused on the selec­ tion of accessions that are quite able to exploit these associated beneficial microbial communities. 1. Introduction Plants through their root system and surrounding soil influenced by root exudates represent an interesting ecological niche for the develop­ ment of soil microbiota which are able to colonize the rhizosphere, roots and eventually move to the above­ground plant parts (Compant et al., 2019). Due to its ecological importance and functional diversity, the rhi­ zosphere microbiome was intensively explored for various features (Marques et al., 2014; Edwards et al., 2015; Gopal and Gupta, 2016; Compant et al., 2019). Microbiotas associated to roots are derived from the soil environment which contains highly diverse microorganisms including Acidobacteria, Verrucomicrobia, Bacteroidetes, Proteobacteria, (*) Corresponding author: raniaaydi@yahoo.fr Citation: AYDI BEN ABDALLAH R., CHIKH­ROUHOU H., JABNOUN­KHIAREDDINE H., DAAMI­REMADI M., 2024 ­ Pumpkins (Cucurbita spp.) diversity and their associated microbiota. ­ Adv. Hort. Sci., 38(1): 13­24. Copyright: © 2024 Aydi Ben Abdallah R., Chikh­Rouhou H., Jabnoun­Khiareddine H., Daami­Remadi M. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 1 November 2022 Accepted for publication 11 January 2024 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-13913 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2024 38(1): 13­24 14 Planctomycetes, and Actinobacteria (Fierer, 2017). Seeds may be colonized by various microorganisms which proliferate later in the roots of the developing plant and colonize the rhizosphere (Compant et al., 2019). Soil microbial communities play key roles in plant development and health (Philippot et al., 2013; Adam et al., 2018). In fact, they may be associated to growth promotion, improved nutrient uptake, and enhanced tolerance to various abiotic and/or biotic stresses (Trivedi et al., 2020). The below­ground microbial composition is influenced by many abiotic and biotic factors including soil traits (pH, salinity, structure, moisture, organic matter, environmental conditions), relative abundance of soilborne bio­ aggressors, plant species, genotypes, and agricultural and disease management practices (Hardoim et al., 2015; Fierer, 2017; Compant et al., 2019). All the above­mentioned factors contribute, at variable degrees, to the definition of the root microbial com­ munity structure together with the host­related fac­ tors like plant age and developmental stage, health status and the composition of root exudates (Bulgarelli et al., 2012). Based on Carelli et al. (2000) investigation, the rhizosphere community composi­ tion varies between plant species and even within the same species between plant genotypes. Also, the root exudates play a key role in recruiting and shap­ ing the soil microbial population structure as they serve as nutrient sources for rhizosphere microor­ ganisms (Sung et al., 2006) and represents an impor­ tant component of communication with rhizosphere­ inhabiting microorganisms (Haichar et al., 2014). Hence, the variation in the chemical composition of root exudates between and within plant species (Grayer et al., 2004) may lead to the development and the proliferation of a phylogenetically diverse array of microorganisms. The chemical composition of root exudates, resulting of different below­ground interactions and factors (soil chemical and physical properties, plant species, age, etc), may impact the soil microbial community structure and function by influencing plant physiology and development (Griffiths et al., 1999). In fact, among the members of the rhizosphere microbiome, some are beneficial for plant growth and resilience but others may be phy­ topathogenic exhibiting capacity to overcome the innate plant defense system and to cause devastating diseases (de Faria et al., 2021). Pumpkin (Cucurbita spp.) is an extraordinary veg­ etable species that may be exploited for medicinal and nutritional features (Tlili et al., 2020; Hosen et al., 2021; Chikh­Rouhou et al. 2023 b). However, pumpkin cultivation is still ignored in some countries. Cucurbita pepo L., C. maxima Duchesne, and C. moschata Duchesne are three pumpkin species eco­ nomically important which are grown over various agricultural regions worldwide (Maynard et al., 2002). In Tunisia, pumpkin has significant economic importance especially as familiar agriculture because of its rusticity, high nutritional values and long post­ harvesting conservation. There is no improved culti­ var in Tunisia and the production of Cucurbita is based on local accessions and landraces. Chikh­ Rouhou et al. (2019, 2023 a, 2023 b) evidenced that pumpkin landraces collected from farmers of the Centre­East of Tunisia belongs to three species namely C. maxima, C. pepo, and C. moschata with a predominance of C. maxima. Pumpkins face a num­ ber of constraints including a shortage of genetically improved seeds, infections with various pests and pathogens (Ndinya, 2019) in addition to the plant parasitic nematode Pratylenchus (Zhao et al., 2022). Developing new cultivars with superior qualities, higher mineral contents, important yield and average weight of fruits, potential resistance towards pests and fungal diseases, tolerance to environmental diffi­ culties, shelf lives enhancement is highly required (Paris, 2016; Seymen et al., 2016; Hosen et al., 2021). Breeding plants for beneficial plant­microbe inter­ actions is an emerging field mainly focusing the below­ground interactions in the rhizosphere and their valorization for the development of economical­ ly and ecologically interesting plant material (Bakker et al., 2012; Adam et al., 2018). In fact, breeding shapes the composition of the root­associated micro­ bial communities including the antagonistic potential towards the encountered pathogens (Peiffer and Ley, 2013; Bouffaud et al., 2014; Cardinale et al., 2015). Thus, breeding strategy is recently focused on geno­ types­microbial holobiont interactions in order to generate diverse new phenotypes without altering plant genomic information (Wei and Jousset, 2017; Adam et al., 2018; Wille et al., 2018). Therefore, this study aimed to select the most productive pumpkin accession among 12 tested, to determine their associated culturable soil microbial community and to search for an eventual link between fruit and yield parameters and their associ­ ated microorganisms and soil traits. Aydi Ben Abdallah et al. ‐ Microbiota associated to pumpkins 15 2. Materials and Methods Plant material Twelve (12) pumpkin (Cucurbita spp.) accessions belonging to three Cucurbita species (C. maxima, C. moschata, and C. pepo) are used in this study. Their main traits are detailed in Table 1 and figure 1. They were obtained from the Cucurbits breeding program at the Regional Research Centre on Horticulture and Organic Agriculture (CRRHAB), Chott­Mariem, Tunisia. For each accession, seeds were sown in cell trays and maintained at 25°C under greenhouse condi­ tions. At the two­true­leaf growth stage, they were further transplanted (end of March) to an open field at the experimental station of CRRHAB of Sahline, Tunisia (N35° 45’05’’, E10°42’39’’). Experimental design Pumpkins seedlings were transplanted into rows with a distance of 120 cm between seedlings within the same row and 80 cm between rows. The trial was conducted under drip irrigation system without inputs. Cattle manure was applied at a rate of 500 Kg ha­1 before planting. The experimental design was a completely randomized block design. Two replicates of six seedlings each were used per each accession tested. Soil sampling Composite soil samples from each replicate were collected at the initial state (before planting) (Table 2) and four times post planting i.e. at 30, 60, 90, 150 days post­planting (DPP). After planting, three soil cores (7 cm in diameter × Table 1 ­ Pumpkin accessions (species and characteristics) used in this study and their main traits Fig. 1 ­ Diversity of Pumpkin (Cucurbita spp.) accessions used in the study (A) and their plant growth habits (B). C7, C15: Cucurbita pepo. C2, C5, C6.2, C9.1, C9.2, C14.2, C15.1, C23: Cucurbita maxima. C14.1, C26: Cucurbita moschata. * Highly susceptible to powdery mildew (data not shown). Accession codes Cucurbita species Plant growth habit Fruit shape Flesh color C2 C. maxima Bushy Transverse broad elliptic Yellow C5 C. maxima Prostrate Medium elliptic Orange C6.2 C. maxima Intermediate Globular Yellowish orange C7 C. pepo Bushy Globular Cream C9.1* C. maxima Prostrate Transverse medium elliptic Yellow C9.2 C. maxima Intermediate Heart shaped Yellowish orange C14.1 C. moschata Intermediate Top shaped Yellowish orange C14.2 C. maxima Prostrate Medium elliptic Yellow C15* C. pepo Bushy Transverse elliptical Yellow C15.1* C. maxima Intermediate Transverse medium elliptic Orange C23 C. maxima Prostrate Transverse elliptical Orange C26 C. moschata Prostrate Transverse broad elliptic Orange Adv. Hort. Sci., 2024 38(1): 13­24 16 15 cm in depth) were removed from the rhizosphere soil of each sampled plant and were combined to make one composite soil per accession. At the initial state (before planting), ten soil cores were removed and were combined to make one composite soil sam­ ple. Two replicates were considered for each soil sampling. Once brought to laboratory, soil samples were passed through a 2­mm sieve to remove rocks and large organic debris. They were stored in plastic bags at 10°C until use. Two subsamples were processed from each soil sample. Determination of soil pH and electrical conductivity (EC) Each composite soil sample was air­dried and sus­ pended into distilled water (1:10 soil H2O ­1 ratio). Soil filtrates obtained by filtration through Whatman paper No. 1 were analyzed for the determination of their pH and electrical conductivity (EC) using a glass electrode (VWR sympHony®) and a digital conductivi­ ty meter (HANNA®), respectively. Estimation of soil microbial community structure General populations of culturable soil microorgan­ isms were determined using the soil dilution plating techniques on various agar media according to Larkin and Honeycutt (2006) with some modifications. For each subsample taken from each composite soil, 10 g were added to 90 ml of sterile 0.2% water agar, vig­ orously stirred for 30 min, serially diluted and a­100 µl sample was plated on 10% Tryptic Soy Agar (TSA) for total bacterial counts, Yeast Malt Agar (ISP medi­ um No. 2) amended with 75 mg l­1 of nalidixic acid and 100 mg l­1 of cyclohexamide for actinomycete counts, and Potato Dextrose Agar (PDA) amended with 300 mg l­1 of streptomycin sulphate for total fungal counts. Three replicates of one plate each were used for each soil subsample. Bacterial and actinomycete plates were incubated at 28°C for 2 and 14 days, respectively, and fungal plates were maintained at 25°C for 7 days. Colonies of Trichoderma spp., Aspergillus spp., and Fusarium spp. were identified based on their macro­ and micro­morphological traits (Barnett and Hunter, 1987) under light microscope and counted separate­ ly. Colony­forming units (CFU) were counted to esti­ mate the microbial density on each selective medium (Marin et al., 2013). The soil microbial population counts were estimated per 1 g of fresh soil. Yield parameters The average fruit weight and the average yield per plant were noted at five months post­planting. The average fruit weight parameter was determined for three randomly sampled plants. Statistical analysis Data were subjected to a one­way analysis of vari­ ance (ANOVA) using Statistical Package for the Social Sciences (SPSS) software for Windows version 16.0. Data for pH, EC of soil samples and rhizosphere microbial population counts were analyzed according to a completely randomized factorial model with two factors (Accessions tested × Sampling times). As for yield parameters, data were analyzed according to a completely randomized block design. Experiments were repeated twice. Means were separated using Tukey test to identify significant pair­wise differences at P≤0.05. Correlations between fruit weight and yield para­ meters and soil characteristics (pH, EC and microbial community structure) were carried out using Pearson’s test at P≤0.05. For an overview of pumpkins accessions distribu­ tion, and to explore soil microbial community con­ tributing to classification, a Principal Component Analysis (PCA) was also performed using SPSS. 3. Results Variation of soil pH and EC ANOVA analysis of pH values varied significantly Table 2 ­ Soil characteristics estimated at the initial state (before pumpkin planting) as determined by soil dilu­ tion (z) plating on selective media (z) Soil sample was a composite soil from twenty soil cores collec­ ted before planting and soil dilution was made from a concentra­ tion of 10% (w v­1). (y) CFU= Colony­Forming Unit. Soil characteristic Data Initial soil characteristics pH 7.42 EC (dS m­1) 0.56 Culturable microbial population (CFU (y) g‐1 fresh Total bacteria (× 107) 2.99 Actinomycetes (× 104) 0.95 Total fungi (× 104) 1.62 Aspergillus spp. (× 103) 0.12 Trichoderma spp. (× 103) 1.12 Fusarium spp. (× 103) 0.18 Aydi Ben Abdallah et al. ‐ Microbiota associated to pumpkins 17 (at P≤0.05) depending on sampling times only. No significant difference was noted between pumpkins accessions and between both factors (Table 3). A sig­ nificant decrease of pH values of about 9.9 to 12.7% was noted at harvest (150 DPP) as compared to soil samples collected at 30, 60 and 90 DPP (Table 3). ANOVA analyses revealed a significant variation in EC values among accessions, sampling times and their interaction (Table 3). The highest EC values were recorded in the rhizopshere of C. maxima C9.1, C. maxima C14.2, C. pepo C7 and C. pepo C15. As for the sampling time effect on this parameter, the EC of the rhizosphere soil associated to the twelve pump­ kins accessions was 34.4­35% and 18.1­19% higher at 30­60 DPP than at 90 and 150 DPP, respectively (Table 3). Variation of the culturable soil microbial structure The number of bacterial and actinomycetes colonies varied significantly (at P≤0.05) among pump­ kins accessions, sampling times and their interaction (Table 4). The highest population of culturable bacte­ ria was obtained from the rhizosphere of C. maxima C23 and C. pepo C15 which was 33.1­55.8% and 15.8­ 44.4% more abundant than those of the remaining accessions (Table 4). The abundance of culturable bacteria in the rhizosphere of all the remaining acces­ sions was significantly comparable. Concerning the effect of the sampling times (all accessions com­ bined) on this parameter, bacterial colonies counts Table 3 ­ pH and electrical conductivity (EC) of soil samples rem­ oved from the rhizosphere of pumpkins depending on accessions tested and sampling times (z) C7 and C15= Cucurbita pepo. C2, C5, C6.2, C9.1, C9.2, C14.2, C15.1 and C23= C. maxima. C14.1 and C26= C. moschata. (y) Accessions means (for all sampling times combined) followed by the same letter are not significantly different according to Tukey test at P≤0.05. (x) Sampling times means (for all accessions combined) followed by the same letter are not significantly different according to Tukey test at P≤0.05. (w) DPP= Days post­planting. Soil samples pH EC (dS m­1) Accessions (z) means (y) C2 7.30 a 0.507 ef C5 7.22 a 0.45 f C6.2 7.27 a 0.61 bc C7 7.18 a 0.63 abc C9.1 7.24 a 0.69 a C9.2 7.24 a 0.508 def C14.1 7.17 a 0.57 cd C14.2 7.21 a 0.66 ab C15 7.14 a 0.62 abc C15.1 6.69 a 0.56 cde C23 7.19 a 0.47 f C26 7.27 a 0.503 ef Sampling times means (x) 30 DPP (w) 7.44 a 0.62 a 60 DPP 7.25 a 0.61 ab 90 DPP 7.48 a 0.4 c 150 DPP 6.53 b 0.5 b Source of variation p­values Accessions (Acc) 0.27 P ≤ 0.001 Sampling times (ST) P≤0.001 P ≤ 0.001 Acc × ST 0.48 P ≤ 0.001 Table 4 ­ Culturable bacterial, actinomycetes and fungal popula­ tion densities in soil samples (CFU g­1 of fresh soil) rem­ oved from the rhizosphere of pumpkins plants depen­ ding on accessions tested and sampling times (z) C7 and C15= Cucurbita pepo. C2, C5, C6.2, C9.1, C9.2, C14.2, C15.1 and C23= C. maxima. C14.1 and C26= C. moschata. (y) Accessions means (for all sampling times combined) followed by the same letter are not significantly different according to Tukey test at P≤0.05. (x) CFU= Colony forming unit. (w) Sampling times means (for all accessions combined) followed by the same letter are not significantly different according to Tukey test at P≤0.05. (v) DPP= Days post­planting. Culturable microbiome population Bacteria Actino­ mycetes Fungi Accessions (z) means (y) CFU (x) g­1 of fresh soil × 108 × 105 × 105 C2 1.68 bc 0.96 bc 1.36 a C5 1.62 bc 1.68 a 0.93 a C6.2 2.24 bc 1.20 abc 1.22 a C7 1.48 c 1.08 bc 1.69 a C9.1 1.59 bc 1.08 bc 1.59 a C9.2 1.80 bc 0.96 bc 1.58 a C14.1 1.77 bc 1.05 bc 1.63 a C14.2 2.04 bc 1.13 abc 1.32 a C15 2.66 ab 0.82 c 1.45 a C15.1 1.98 bc 0.90 c 1.28 a C23 3.35 a 1.53 ab 1.02 a C26 1.93 bc 0.74 c 1.20 a Sampling times means (w) CFU g­1 of fresh soil × 108 × 105 × 105 30 DPP (v) 3.15 a 0.93 b 1.57 ab 60 DPP 3.08 a 2.86 a 1.86 a 90 DPP 1.06 b 0.27 c 1.41 b 150 DPP 0.74 b 0.32 c 0.58 c Sources of variation p­values Accessions (Acc) P≤0.001 P≤0.001 0.06 Sampling times (ST) P≤0.001 P≤0.001 P≤0.001 Acc × ST P≤0.001 P≤0.001 0.15 18 Adv. Hort. Sci., 2024 38(1): 13­24 from the rhizosphere of all pumpkins accessions noted at 30 and 60 DPP were 65.6­66.3 and 75.9­ 76.5% significantly higher than those recorded at 90 and 150 DPP, respectively. Actinomycetes community was abundant on the rhizopshere of C. maxima C5, C. maxima C6.2, C. maxima C14.2 and C. maxima C23 which was 35.7­ 55.9%, 10­38­3%, 4.4­34.5% and 29.4­51.6% higher than that associated to the remaining accessions. For all pumpkins accessions combined, the actino­ mycetes population was 67.5, 90.5 and 91.9% signifi­ cantly higher at 60 DPP than at 30, 90 and 150 DPP, respectively. Data given in Table 4 showed that the total cultur­ able fungal community varied significantly (at P≤0.05) depending on sampling times only and that all acces­ sions tested exhibited significantly comparable fungal community populations. Fungal colonies recovered from the rhizosphere of all pumpkins accessions at 60 DPP were 15.6, 24.2 and 68.8% significantly higher than those recovered at 30, 90 and 150 DPP, respec­ tively. As for fungal community structure, culturable Aspergillus spp. and Trichoderma spp. populations varied significantly (at P≤0.05) in the rhizosphere of pumpkins plants depending on tested accessions, sampling times and their interaction (Table 5). For instance, the rhizospheric Aspergillus spp. communi­ ty associated to C. maxima C14.2 was significantly 40­48.8% more abundant than that associated to C. moschata C26 and C. maxima C2 accessions. Furthermore, Trichoderma spp. population was sig­ nificantly 75.9­84.1% higher at the rhizosphere of C. maxima C14.2 than at that of C. pepeo C7 and C. Table 5 ­ Culturable fungal population structure in soil samples (CFU g­1 of fresh soil) removed from the rhizosphere of pumpkins plants depending on accessions tested and sampling times +(z) C7 and C15= Cucurbita pepo. C2, C5, C6.2, C9.1, C9.2, C14.2, C15.1 and C23= C. maxima. C14.1 and C26= C. moschata. (y) Accessions means (for all sampling times combined) followed by the same letter are not significantly different according to Tukey test at P≤0.05. (x) CFU= Colony forming unit. (w) Sampling times means (for all accessions combined) followed by the same letter are not significantly different according to Tukey test at P≤0.05. (v) DPP= Days post­planting. Culturable fungal population Aspergillus spp. Trichoderma spp. Fusarium spp. Accessions (z) means (y) CFU (x) g­1 of fresh soil × 104 × 104 × 104 C2 1.92 b 1.33 abc 0.07 a C5 3 ab 2 ab 0.83 a C6.2 3.08 ab 0.66 abc 1.5 a C7 3.33 ab 0.50 bc 0.07 a C9.1 3.25 ab 0.33 c 0.83 a C9.2 3 ab 0.83 abc 1.66 a C14.1 2.42 ab 1.50 abc 0.08 a C14.2 3.75 a 2.08 a 0.09 a C15 2.58 ab 1.58 abc 0.08 a C15.1 2.83 ab 1.17 abc 0.07 a C23 2.75 ab 1.75 abc 0.09 a C26 2.25 b 1.17 abc 0.08 a Sampling times means (w) CFU g­1 of fresh soil × 104 × 104 × 104 30 DPP (v) 0.83 c 1.08 ab 0.27 a 60 DPP 0.55 c 0.72 b 0.08 a 90 DPP 1.44 b 1.58 a 1.38 a 150 DPP 9.81 a 1.58 a 0.27 a Sources of variation p­values Accessions (Acc) P ≤ 0.01 P ≤ 0.01 0.14 Sampling times (ST) P ≤ 0.001 P ≤ 0.01 0.68 Acc × ST P ≤ 0.001 P ≤ 0.001 0.35 Aydi Ben Abdallah et al. ‐ Microbiota associated to pumpkins 19 maxima C9.1 (Table 5). Aspergillus spp. colonies recovered from the rhi­ zosphere of all pumpkins accessions at 150 DPP were 85.3 and 94.4% significantly higher than those recov­ ered at 90 and 30­60 DPP, respectively. Trichoderma spp. population estimated was significantly higher (+54.4%) at 150 and 90 DPP than at 60 DPP. Concerning Fusarium spp. populations, no significant differences were detected between pumpkins acces­ sions and sampling times nor their interaction (Table 5). Variation of fruit production and yield among pump‐ kins accessions tested Analysis of variance revealed a significant (at P≤0.05) variation of the average fruit weight between the pumpkin accessions. The highest average fruit weights ranging between 4.18 and 8.48 Kg were noted in the accessions C5, C14.2, C15.1 and C23 of C. maxima and C14.1 of C. moschata whereas for the remaining seven pumpkins accessions, this parame­ ter varied between 2.53 and 3.9 Kg (Fig. 2A). The average fruit yield produced per plant varied significantly (at P ≤ 0.05) among pumpkins acces­ sions. Four C. maxima accessions (namely C5, C6.2, C14.2, and C23) and one C. moschata accession (C26) produced significantly the highest fruit yields per plant (5.31­6.21 Kg plant­1) than the remaining ones (0.91­3.96 Kg plant­1) (Fig. 2B). Correlation between production and yield parameters and soil characteristics Pearson’s correlation analysis indicated that the average fruit weight was significantly and positively correlated to the associated actinomycetes commu­ nity (r= 0.765, P= 0.004) and Trichoderma spp. popu­ lation (r= 0.697, P= 0.012) but it was significantly and negatively (r= ­0.700, P= 0.01) linked to the total cul­ turable fungal population in the analyzed soil sam­ ples (Fig. 3). Pearson correlation analysis, also, revealed a sig­ nificant and negative correlation between the aver­ age fruit yield per plant and the fungal population (r = ­0.701; P = 0.011) colonizing the rhizosphere of pumpkins accessions (Fig. 3). Multicriteria analysis via PCA Based on the PCA analysis performed, the first two main components (PC) comprised about 68.31% of the variability existing in the analyzed genotypes. PC­1 explained 47.05% of the total variability. The most important traits related to this axis were: the fruit fresh weight, the yield per plant, and actino­ mycetes and Trichoderma spp. population. The most important traits of PC­2, which explained 21.25% of the total variation, were EC values and Aspergillus spp. community (Fig. 4A). The distribution of pumpkins accessions among the two axes showed the variability and allowed dis­ tinguishing 3 main groups (Fig. 4B). The 1st group included 4 accessions (C5, C23, C14.2 and C6.2 belonging to C. maxima) characterized by the highest average fruit weight, the highest yield per plant, the Fig. 2 ­ A, B Average fruit weight and yield per plant of pumpkins accessions noted five months post­planting. Bars sharing the same letter are not significantly different according to Tukey test at P ≤ 0.05. The average fruit weight (A) and the average yield (B) per plant were determined at har­ vest. C7 and C15: Cucurbita pepo. C2, C5, C6.2, C9.1, C9.2, C14.2, C15.1, and C23: C. maxima. C14.1 and C26: C. moschata. Fig. 3 ­ Heat map of Pearson's correlation (r) between the aver­ age fruit weight and the average yield per plant of pump­ kins accessions and soil characteristics. Asterisks indicate statistically significant correlation values, negative or positive at * P≤0.05 and ** P≤0.01. Adv. Hort. Sci., 2024 38(1): 13­24 20 highest actinomycetes and bacterial communities, the highest Trichoderma spp. and Aspergillus spp. populations, and the lowest fungal community in their rhizosphere. The 2nd group was comprised of C7 (C. pepo) and C9.1 (C. maxima) accessions character­ ized by the lowest fruit weight and the lowest Trichoderma spp. populations, and the 3rd group was composed of the remaining 6 accessions exhibiting intermediate yield per plant and Trichoderma spp. populations. 4. Discussion and Conclusions Plant­associated microbiome plays a fundamental role in plant growth and health (Wei and Joussset, 2017). Breeding programs focusing genotype­associ­ ated beneficial microbiome help achieve ecologically desired plant phenotype traits (Adam et al., 2018; Wille et al., 2018). The current study aimed to select the most productive pumpkins accessions based on the variability of their soil microbial community struc­ ture and to investigate the presence of eventual links between Cucurbita spp. production and yield para­ meters and their rhizosphere soil associated microor­ ganisms. Our results clearly demonstrated that Cucurbita species and accessions shaped their own soil microbial community structure. Some microbes have a particular affinity for certain pumpkins acces­ sions in determining rhizosphere communities. The variation of composition of microbial distribution in the rhizosphere of Cucurbita spp. accessions may be explained by the differences in their root morphology and the composition and content of their root exu­ dates which play a fundamental role in the recruit­ ment of plant holobiont. Plant­associated microbio­ me and their interactions are highly diverse and mul­ tiple factors shape the microbial community assem­ bly and functioning. In fact, the microbial communi­ ty’s structure varies significantly depending on plant species and/or genotypes growing in the same soil environment (Kang and Mills, 2004; Yao and Wu, 2010; Berendsen et al., 2012; Aydi Ben Abdallah et al., 2023) and even on plant growth stage (Chaparro et al., 2014; Compant et al., 2019). The variation in the soil­associated microbiome communities has been assigned to the differences in the root morphol­ ogy, the type of rhizodeposits, the amount and the composition of root exudates and mainly carbon sources which are limiting factors for microbial activi­ ty and proliferation (Marschner et al. , 2007; Broeckling et al., 2008; Compant et al., 2019). Moreover, edaphic factors such as soil pH, electrical conductivity (EC), soil texture, soil parental material, and soil salinity are important determinants of com­ munity structure and diversity of soil microbiome (Lozupone and Knight, 2007; Lauber et al., 2008; Xu et al., 2014; Sun et al., 2015; Min et al., 2016). The soil­associated microbiomes have an effect on plant growth and yield production. Positive and sig­ nificant correlations were determined between fruit fresh weight and the culturable bacterial and Trichoderma spp. populations in the rhizopshere of pumpkins accessions tested in the current investiga­ Fig. 4 ­ PCA biplot the variability existing in the analyzed traits (A) and the distribution of pumpkins accessions (B). Ec: Electrical conduc­ tivity. Fung: Fungi. Asp: Aspergillus spp. Yield: Yield per plant. Act: Actinomycetes. Fweight: Fruit weight. Bact: Bacteria. Tric: Trichoderma spp. C7 and C15: Cucurbita pepo. C2, C5, C6.2, C9.1, C9.2, C14.2, C15.1 and C23: C. maxima. C14.1 and C26: C. moschata. Aydi Ben Abdallah et al. ‐ Microbiota associated to pumpkins 21 tion. Plant­associated microbes with their plant growth­promoting traits play a crucial role in enhanc­ ing plant biomass and crop yield (Kumar et al., 2022). Halifu et al. (2019) demonstrated that inoculation with two Trichoderma species on Pinus Sylvestris var. mongolica seedlings had a positive correlation with growth parameters, soil nutrient content, and soil enzymatic activity in their rhizosphere. Trichoderma spp. are able to increase the growth and the exten­ sion of the root system and to stimulate the secre­ tion of extracellular enzymes such as sucrase, urease, phosphatase, and organic acids in the rhizosphere. These compounds lead to the improvement of the nutrient cycle and the soil enzymatic activity and con­ sequently the soil nutrient status and availability (Pelagio­Flores et al. , 2017). Furthermore, Trichoderma spp. can secrete the indole 3 acetic acid (IAA) and to promote the growth of many crops as previously demonstrated for cucumber, bottle gourd, and bitter gourd (Kotasthane et al. , 2015). Furthermore, the volatile and non­volatile secondary metabolites released by Trichoderma spp. such as 6­ n­pentyl­6H­pyran­2­one (6PP), gliotoxin, viridin, harzianopyridone, harziandione, and peptaibols have a significant growth­promoting effect on plants (José et al., 2008). Mohanty et al. (2021) also demonstrat­ ed that the beneficial bacterial communities may improve crop productivity as part of sustainable agri­ culture. In fact, Acidothiobacillus ferooxidans and Bacillus cereus are associated to increased growth and yield and improved soil composition in pumpkin (Ansari et al., 2017). Plant growth improvement may be achieved either directly via the enhancement of nutrient avail­ ability and phytohormone modulation and/or indi­ rectly through the biocontrol activity i.e. suppression of associated pathogens and/or the alleviation of biotic and abiotic stresses leading to the improve­ ment of both plant health and crop productivity (Khan et al., 2020; Basu et al., 2021; Zhang et al., 2021; Kumar et al., 2022). In the current study, nega­ tive and significant correlations were noted between the average fruit weight and yield per plant parame­ ters and the total culturable fungal populations. The fungal population estimated in the rhizosphere of pumpkins accessions may be mainly composed of soilborne pathogens naturally associated to pump­ kins plants which may be involved in the recorded decreases in fruit weight and yield. Based on ACP analyses, the 1st group was comprised of 4 accessions of C. maxima (namely C5, C23, C14.2 and C6.2) which are characterized with the highest production para­ meters (average fruit weight and yield per plant) and the highest populations of actinomycetes, bacteria, Trichoderma spp. and Aspergillus spp., and also the lowest fungal community in their rhizosphere. Hence, these four microbial groups (bacteria, Aspergillus spp., Trichoderma spp. and actinomycetes) predomi­ nant in the rhizosphere of these 4 most productive pumpkins accessions may be involved, either individ­ ually or in consortium, indirectly in the promotion of pumpkins yield via their eventual antagonistic poten­ tial against their associated fungal pathogens. As demonstrated in Yang et al. (2017) study, some potential plant­beneficial microbial agents could act as network key, thus reducing the chance of a given a soil­borne pathogen to invade the target plant species. Also, Chaurasia et al. (2018) demonstrated the successful role of actinomycetes on plant protec­ tion and growth promotion of Solanaceae, Cucurbitaceae, Brassicaceae, Amaranthaceae, Umbelliferous, Asteraceae, Fabaceae, Asparagaceae, and Amaryllidaceae vegetable crops. Also, as demon­ strated in Hung and Rutgers (2016) study, Aspergillus spp. are multifaceted fungi that the plant benefits with different manner such as plant growth promo­ tion and protection. Pascual et al. (2017) also empha­ sized the role of T. harzianum in reducing the natural infection of melon plants by F. oxysporum f. sp. melo‐ nis and in improving their yields. In Aydi Ben Abdallah et al. (2019) study, Bacillus subtilis SV41 and B. amy‐ loliquefaciens subsp. amyloliquefaciens SV65 have successfully decreased the soil infection potential by Fusarium species, suppressed Fusarium wilt severity and enhanced tomato growth and production. In our study, the 4 selected pumpkins accessions are quite able to exploit their associated beneficial indigenous microbial communities which could be considered in the future pumpkin breeding pro­ grams. In conclusion, this study clearly demonstrated the significant role of tested accessions in affecting the distribution of microbial community in their rhizos­ phere leading to differences in yield parameters between pumpkins accessions. The variation in the microbial community structure with the accessions tested might be due to the changes in the composi­ tion of their root exudates which need to be more elucidated in our future investigations. Four acces­ sions of C. maxima (namely C5, C23, C14.2 and C6.2) have a great potential as they are characterized by the highest average fruit weight and yield per plant, Adv. Hort. Sci., 2024 38(1): 13­24 22 the highest populations of actinomycetes, bacteria, Trichoderma spp. and Aspergillus spp., and the low­ est fungal population in their rhizosphere. Thus, the exploitation and the re­integration of the recovered beneficial bacterial and Trichoderma spp. populations associated with these four selected accessions of C. maxima will be considered in the future pumpkin breeding programs to reduce the threat imposed by their soil­borne pathogens and consequently led to more enhancements in pumpkin fruit yield into the less productive accessions. Acknowledgements This work was funded by the Ministry of Higher Education and Scientific Research of Tunisia through the funding allocated to the research laboratory LR21AGR03­Production and Protection for a Sustainable Horticulture (2PHD), IRESA­University of Sousse, Regional Research Centre on Horticulture and Organic Agriculture of Chott­Mariem, Tunisia. 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