Figures (2)  Tables (3)

    • Figure 1. 

      Relative abundance of genera of endophytic fungi and the set of epiphytic and endophytic fungi associated with the plant material from the 16 almond cultivars analysed.

    • Figure 2. 

      Evolution of interactions on potato dextrose agar between Colletotrichum godetiae and fungal antagonists obtained from almond trees, 4, 8, and 11 days after inoculation (dai). The fungal isolates to be tested are located at the top of the Petri dishes, while Colletotrichum godetiae (Cg) is located at the bottom. Excessive growth of (a) Neurospora intermedia mycelium on C. godetiae is observed; excessive growth of (b) Neopestalotiopsis elaeagni on C. godetiae by contact inhibition, without the formation of a visible inhibition zone; overgrowth of (c) Epicoccum italicum mycelium on C. godetiae, creating a dividing line without contact between the colonies; C. godetiae mycelium is replaced by (d) Clonostachys chloroleuca; the growth of (e) Talaromyces amestolkiae and C. godetiae stops at the contact line of the mycelium of both colonies, causing a reddish discolouration at the contact edge of C. godetiae; (f) Colletotrichum godetiae in the control culture.

    • Cultivar/Organ Endophytes Endophytes and epiphytes
      No. of isolates No. of genera FC (%) H No. of isolates No. of genera H
      Cultivar Belona 1,119 21 75.9 2.14 355 17 1.64
      Carmel 588 23 74.4 2.31 202 15 2.10
      Casa Nova 67 8 44.7 1.65 83 12 1.97
      Constantine 216 15 62.4 2.07 130 14 1.76
      Ferragnes 31 6 32.3 1.72 65 10 1.79
      Ferralise 32 8 26.7 1.56 88 9 1.46
      Francoli 31 5 47.0 1.11 35 11 2.12
      Guara 684 23 50.1 2.05 680 18 1.85
      Lauranne 2,701 26 54.0 2.34 2,303 22 2.19
      Marinada 813 20 64.4 2.23 449 15 1.96
      Marta 274 16 61.0 2.18 175 10 1.82
      Monterey 741 22 64.0 2.41 416 15 2.14
      Nonpareil 700 21 68.6 2.42 321 17 2.28
      Penta 399 16 67.4 2.13 193 12 1.90
      Shasta 314 12 74.4 2.13 108 6 1.27
      Soleta 2,195 28 67.1 2.25 1,078 22 1.96
      Vairo 614 17 49.4 2.23 630 18 2.09
      Wood Colony 693 22 70.9 2.42 284 11 1.98
      Organ Fruits 3,576 28 92.5 2.15 290 16 2.21
      Leaves 2,504 27 63.1 2.25 1,463 19 2.21
      Flowers 2,605 26 43.7 2.13 3,354 28 2.09
      Branches 3,527 33 58.6 2.18 2,488 27 2.20
      The results are presented separately for endophytic communities and for the combined endophytes and epiphytes communities. FC: Frequency of colonization (%); H: Shannon-Wiener index.

      Table 1. 

      Number of isolates, number of genera, colonisation frequency and Shannon-Wiener diversity index (H) of fungi obtained from 16 cultivars and their plant material of Prunus dulcis

    • Isolate code ITS GenBank Accession No. Origin of isolates Family Species
      Location, cultivar and plant material Treatment1
      Dothideomycetes-Cladosporiales
      MR101 PX249825 Ferreira do Alentejo, 'Marinada'
      Flower      		 D Cladosporiaceae Cladosporium sp.
      Dothideomycetes-Dothideales
      MR110 PX250994 Aljustrel, 'Belona', Flower ND Saccotheciaceae Aureobasidium pullulans
      Dothideomycetes-Pleosporales
      MR130 PX251240 Alfândega da Fé, 'Casa Nova', Flower ND Didymellaceae Epicoccum italicum
      MR090 PX249718 Ferreira do Alentejo, 'Francoli', Branch D Epicoccum layuense
      MR106 PX250333 Évora, 'Soleta', Branch ND Nothophoma spiraeae
      MR141 PX251299 Alvito, 'Nonpareil', Leaf D Pleosporaceae Alternaria sp.
      MR104 PX249852 Aljustrel, 'Soleta', Leaf D Alternaria vitis
      MR098 PX249723 Aljustrel, 'Soleta', Flower ND Stemphylium vesicarium
      Eurotiomycetes-Eurotiales
      MR117 PX251002 Beja, 'Soleta', Flower D Aspergillaceae Aspergillus ochraceus
      MR108 PX250988 Ferreira do Alentejo, 'Marinada', Flower ND Aspergillus tubingensis
      MR092 PX249719 Mirandela, 'Ferralise', Leaf ND Penicillium expansum
      MR149 PX251741 Aljustrel, 'Nonpareil', Almond D Trichocomaceae Talaromyces amestolkiae
      Sordariomycetes-Diaporthales
      MR099 PX249724 Macedo de Cavaleiros, 'Ferragnes', Flower ND Diaporthaceae Diaporthe foeniculina
      MR105 PX249856 Ferreira do Alentejo, 'Marinada', Flower ND Cytosporaceae Cytospora italica
      Sordariomycetes-Hypocreales
      MR153 PX251777 Beja, 'Penta', Almond D Bionectriaceae Clonostachys chloroleuca
      MR142 PX395649 Aljustrel, 'Soleta', Branch D Hypocreaceae Trichoderma citrinoviride
      MR093 PX249720 Ferreira do Alentejo, 'Soleta', Branch ND Trichoderma viridescens
      MR089 PX249569 Aljustrel, 'Soleta', Almond D Nectriaceae Fusarium chlamydosporum species complex
      MR127 PX251237 Aljustrel, 'Nonpareil', Branch ND Fusarium tricinctum species complex
      MR134 PX251287 Idanha-a-Nova, 'Guara', Almond D Fusarium annulatum
      MR094 PX249721 Portel, 'Lauranne', Branch ND Fusarium sambucinum species complex
      Sordariomycetes-Sordariales
      MR126 PX251117 Mirandela, 'Vairo', Leaf ND Sordariaceae Neurospora intermedia
      Sordariomycetes-Amphisphaeriales
      MR096 PX249722 Beja, 'Soleta', Flower D Apiosporaceae Apiospora kogelbergensis
      MR137 PX251290 Beja, 'Soleta', Leaf D Sporocadaceae Neopestalotiopsis elaeagni
      MR007 PX251788 Beja, 'Belona', Almond ND Glomerellaceae Colletotrichum godetiae
      1Treatment of plant material: D–Plant material disinfected with 1% NaClO; ND–Plant material not disinfected.

      Table 2. 

      List of 24 isolates used in this study (and the almond anthracnose pathogen), according to their provenance and identification based on a match of the ITS sequence using a phylogenetic tree constructed using the maximum likelihood method

    • Family Species Isolate code Macroscopic analysis1 Average
      colony
      inhibition (%)      		 Colony inhibition
      last day (%)      		 Conidia production (conidia/cm2) Conidia production inhibition (%)
      per unit area      		 Conidia production
      (total of conidia)      		 Conidia production inhibition (%) per total
      Cladosporiaceae Cladosporium sp. MR101 1 −1.84 ± 2.2 gh2 −12.54 ± 4.1 g2 1.27 × 106 ± 7.8 × 104g2 −91.53 ± 11.8 b2 3.89 × 107 ± 1.3 × 106 h2 −455.69 ± 19.0 c2
      Saccotheciaceae Aureobasidium pullulans MR110 1 4.05 ± 2.2 fg 21.07 ± 4.1 eh 4.38 × 105 ± 7.8 × 104 bcdef 33.78 ± 11.8 a 1.26 × 107 ± 1.3 × 106 g −79.79 ± 19.0 a
      Didymellaceae Epicoccum italicum MR130 2, 3 26.83 ± 2.3 cd 32.53 ± 5.3 def 1.78 × 105 ± 5.5 × 104 abc 73.11 ± 8.3 a 1.81 × 106 ± 9.4 × 105 abc 74.12 ± 13.5 a
      Epicoccum layuense MR090 2, 3 19.06 ± 2.2 de 24.28 ± 4.1 ef 5.55 × 105 ± 7.8 × 104 def 16.11 ± 11.8 a 1.05 × 107 ± 1.3 × 106 fg −49.68 ± 19.0 a
      Nothophoma spiraeae MR106 2 −8.54 ± 2.0 h 0.21 ± 4.1 gh 3.83 × 105 ± 7.8 × 104 abcdef 42.17 ± 11.8 a 9.14 × 106 ± 1.3 × 106 dfg −30.44 ± 19.0 a
      Pleosporaceae Alternaria sp. MR141 1 35.34 ± 3.1 bc 53.88 ± 9.3 bcde 3.80 × 105 ± 7.8 × 104 abcdef 42.60 ± 11.8 a 2.62 × 106 ± 1.3 × 106 abcd 62.56 ± 19.0 a
      Alternaria vitis MR104 2 4.30 ± 1.9 fg 10.06 ± 4.1 fgh 3.32 × 105 ± 7.8 × 104 abcdef 49.83 ± 11.8 a 7.67 × 106 ± 1.3 × 106 cdfg −9.56 ± 19.0 a
      Stemphylium vesicarium MR098 2 22.87 ± 2.2 cde 29.38 ± 4.1 ef 2.56 × 104 ± 2.1 × 105 a 96.13 ± 31.2 a 5.35 × 105 ± 3.5 × 106 ab 92.37 ± 50.3 a
      Aspergillaceae Aspergillus ochraceus MR117 1 2.71 ± 2.8 fgh 8.32 ± 4.6 fgh 4.75 × 105 ± 7.8 × 104 cdef 28.27 ± 11.8 a 6.93 × 106 ± 1.3 × 106 bcdfg 1.02 ± 19.0 a
      Aspergillus tubingensis MR108 2 24.50 ± 1.6 cd 37.05 ± 4.1 cde 5.95 × 105 ± 5.5 × 104 def 10.16 ± 8.3 a 8.98 × 106 ± 9.4 × 105 dfg −28.12 ± 13.5 a
      Penicillium expansum MR092 2 32.30 ± 2.2 c 39.29 ± 4.1 cde 1.61 × 106 ± 7.8 × 104 g −143.12 ± 11.8 b 2.86 × 107 ± 1.3 × 106 e −307.76 ± 19.0 b
      Trichocomaceae Talaromyces amestolkiae MR149 1 12.32 ± 2.5 ef −2.70 ± 4.1 g 3.85 × 104 ± 5.5 × 104 a 94.18 ± 8.3 a 4.27 × 105 ± 9.4 × 105 a 93.90 ± 13.5 a
      Diaporthaceae Diaporthe foeniculina MR099 1, 3 21.82 ± 2.2 cde 31.96 ± 4.1 ef 1.40 × 106 ± 7.8 × 104 g −111.29 ± 11.8 b 2.62 × 107 ± 1.3 × 106 e −274.41 ± 19.0 b
      Cytosporaceae Cytospora italica MR105 1, 3 24.59 ± 2.2 cd 39.12 ± 4.1 cde 6.55 × 105 ± 7.8 × 104 ef 0.98 ± 11.8 a 1.05 × 107 ± 1.3 × 106 fg −49.39 ± 19.0 a
      Bionectriaceae Clonostachys chloroleuca MR153 4 9.75 ± 4.2 efg 15.85 ± 9.3 efgh 5.03 × 104 ± 7.8 × 104 ab 92.40 ± 11.8 a 6.34 × 105 ± 1.3 × 106 ab 90.95 ± 19.0 a
      Hypocreaceae Trichoderma citrinoviride MR142 3 77.21 ± 2.5 a 73.30 ± 4.1 ab 1.60 × 105 ± 7.8 × 104 abc 75.87 ± 11.8 a 5.52 × 105 ± 1.3 × 106 ab 92.12 ± 19.0 a
      Trichoderma viridescens MR093 3 83.69 ± 2.2 a 91.61 ± 4.1 a 2.84 × 104 ± 2.1 × 105 a 95.71 ±31.2 a 4.83 × 104 ± 3.5 × 106 a 99.31 ± 50.3 a
      Nectriaceae Fusarium chlamydosporum SC MR089 1, 3 27.83 ± 2.2 cd 42.34 ± 4.1 cde 2.76 × 105 ± 1.2 × 105 abcde 58.31 ± 18.0 a 3.88 × 106 ± 2.0 × 106 abcdf 44.62 ± 29.1 a
      Fusarium tricinctum SC MR127 1 −1.30 ± 3.2 fgh 4.21 ± 5.3 fgh 2.46 × 105 ± 7.8 × 104 abcd 62.91 ± 11.8 a 3.88 × 106 ± 1.3 × 106 abcdf 44.60 ± 19.0 a
      Fusarium annulatum MR134 1, 3 31.27 ± 2.5 c 26.95 ± 4.6 ef 2.88 × 105 ± 7.8 × 104 abcde 56.52 ± 11.8 a 2.48 × 106 ± 1.3 × 106 abcd 64.61 ± 19.0 a
      Fusarium
      sambucinum SC      		 MR094 1, 3 47.52 ± 2.2 b 58.71 ± 4.1 bc 1.34 × 105 ± 7.8 × 104 abc 79.79 ± 11.8 a 1.47 × 106 ± 1.3 × 106 abc 79.02 ± 19.0 a
      Sordariaceae Neurospora intermedia MR126 3 81.81 ± 2.5 a 88.53 ± 4.1 a 1.64 × 105 ± 7.8 × 104 abc 75.15 ± 11.8 a 2.60 × 105 ± 1.3 × 106 a 96.30 ± 19.0 a
      Apiosporaceae Apiospora kogelbergensis MR096 1, 3 24.17 ± 2.2 cde 42.29 ± 4.1 cde 9.92 × 104 ± 9.2 × 104 abc 85.01 ± 13.9 a 1.46 × 106 ± 1.6 × 106 abc 79.12 ± 22.5 a
      Sporocadaceae Neopestalotiopsis elaeagni MR137 1, 3 49.95 ± 2.8 b 58.46 ± 4.6 bcd 2.64 × 105 ± 7.8 × 104 abcde 60.06 ± 11.8 a 1.51 × 106 ± 1.3 × 106 abc 78.47 ± 19.0 a
      Glomerellaceae Colletotrichum godetiae MR007 − − − 6.62 × 105 ± 5.6 × 104 f − 7.00 × 106 ± 9.5 × 105 cdfg −
      Each column shows the mean values ± SE. Average colony inhibition represents the mean inhibition across all assay days and is provided as a descriptive summary of the overall performance of each isolate. 1Macroscopic analysis: (1) The growth of the mycelium of both fungal colonies stops at the contact line; (2) Neither colony grows in the area occupied by the other; (3) Rapid growth of the mycelium of the fungus antagonistic to C. godetiae is observed; (4) Replacement of the mycelium of C. godetiae by the antagonistic fungus. 2In each column, the mean values followed by the same letter are not significantly different according to Tukey test (p < 0.05), which was conducted following the verification of significant differences in the ANOVA for each parameter: Average colony inhibition (%) – F(23,1099) = 116, p-value = 2 × 10−16; Colony inhibition last day (%) – F(23,81) = 40.27, p-value = 2 × 10−16; Conidia production (conidia/cm2) – F(26,612) = 27.26, p-value = 2 × 10−16; Conidia production inhibition (%) per unit area – F(27,610) = 27.20, p-value = 2 × 10−16; Conidia production (total of conidia) – F(26,612) = 31.34, p-value = 2 × 10−16; Conidia production inhibition (%) total – F(27,610) = 38.10, p-value = 2 × 10−16. For the parameter 'Average colony inhibition (%)', Tukey comparisons were applied to the time-averaged values for descriptive purposes, complementing the two-factor ANOVA (Isolate × dpi) presented in the Results section: F(23,1099) = 116, p-value = 2 × 10−16.

      Table 3. 

      Antagonistic effect of 24 fungal isolates on the mycelial growth and sporulation of Colletotrichum godetiae