Results

Pathogenicity tests

To determine the pathogenicity of the Botryosphaeriaceae species identified in this study, two inoculation trials were conducted with six-month-old potted plants of Eucalyptus globulus, E. nitens and Quercus suber. The Q. suber plants were produced from seed collected from a healthy tree. Selected seeds with no signs of fungi or insects were sown in sterile sand and, after germination, potted in a mixture of 2/3 soil and 1/3 sand. The eucalypt plants were obtained from a commercial nursery. In a first trial, Diplodia corticola, D. quercivora and Dothiorella sarmentorum were inoculated onto Quercus suber. In a second trial, Neofusicocum parvum, N. eucalyptorum and Diplodia corticola were inoculated onto the three hosts.

Mycelial plugs of about 2 mm² were cut from the margins of active growing cultures and inserted in a small wound made on the stem 3 cm above the soil. The wounds were covered with sterile moist cotton plugs and wrapped in Parafilm©. On the control plants, the same procedure was applied with a sterile agar plug.

For pathogenicity evaluation, a five levels rating was used based on visual observation of the symptoms, as follows: A = plant without any type of symptom; B = less than ¼ of leaves dry; C = half of the leaves dry; D = more than half of the leaves dry and E = completely dry plant. Symptoms were observed 40 days after inoculation.

The variables resulting from the observations were of the ordinal categorical type and since the samples were small and of variable size, between n = 8 and n = 20, the non-parametric Kruskal-Wallis test was used, which is appropriate for this type of data (Zar 2010).

However, according to Mehta and Patel (1997), when dealing with small and unequal sample sizes, statistical methods based on the asymptotic theory may not be valid and the “p” values calculated by these and the exact methods may be quite different and lead to contradictory results (Mehta and Patel 2010). For this reason, exact tests of complete permutations of the Kruskal-Wallis statistics were used, using the statistical package Statxact® 11 (Cytel Software Corporation© 2015).

Reisolations from the symptomatic inoculated plants were made following the same procedures used on samples, as described above.

Phylogeny

Morphological assignment of strains to different genera were confirmed by ITS BLAST searches on GenBank (Karlin and Altschul 1990). The phylogenetic analysis was performed separately for each genus. The number of sequences and characters in each alignment and the substitution models selected for Bayesian analysis are indicated in table 3.3.

Table 3.3 Statistics and substitution models used in the phylogenetic analysis.

Analysis Figure number

Number of ingroup sequences

Substitution models

Number of unique

sites pattern

Number of characters in alignment

ITS tef1 ITS tef1 ITS tef1 Concatenated

Botryosphaeria 3.4 22 GTR+I HKY+G 95 133 565 304 869

Diplodia 3.5 28 GTR+I+G GTR+I+G 166 139 566 264 830

Dothiorella 3.6 53 GTR+I+G GTR+I+G 176 - 547 - -

Neofusicoccum 3.7 69 GTR+I+G HKY+G 179 198 542 332 874

One isolate from eucalypts, PE 26 grouped with the ex-type strain of Botryosphaeria dothidea (Fig. 3.4). Of the other isolates from eucalypts, six grouped in a well-supported clade containing the ex-type strain of E. eucalyptorum and the remaining five strains, in a clade containing the ex-type strain of N. parvum (Fig. 3.5) and several other species, most of them recently synonymized with N. parvum (Zhang et al. 2021). Fifteen isolates obtained from Quercus suber clustered in a clade with the type strain of D. corticola, three with the ex-type of Diplodia quercivora (Fig. 3.6) and the remaining two isolates within a clade containing several species, most of them recently synonymized with Dothiorella sarmentorum (Zhang et al. 2021) (Fig. 3.7).

Figure 3.4 Phylogenetic tree of Botryosphaeria species resulting from a maximum likelihood (ML) analysis of combined ITS and tef1 datasets. Maximum likelihood bootstrap support values (BS ≥ 50 %) and Bayesian posterior probabilities (PP ≥ 0.85) are shown at the nodes (BS/PP). Ex-type strains are indicated in bold font and strains identified in this study are shown in red. Names in round parentheses are synonyms. The bar represents 0.02 substitutions per site. The tree was rooted to Cophinforma eucalypti MFLUCC 11-0425.

Figure 3.5 Phylogenetic tree of Neofusicoccum species resulting from a maximum likelihood (ML) analysis using combined ITS and tef1 gene region datasets. Maximum likelihood bootstrap support values (BS ≥ 50 %) and Bayesian posterior probabilities (PP ≥ 0.85) are shown at the nodes (BS/PP). Ex-type strains are indicated in bold font and strains identified in this study are shown in red. Names in round parentheses are synonyms. The bar represents 0.05 substitutions per site. The tree was rooted to Botryosphaeria corticis CBS 119047 and B.

dothidea CBS 115476.

Figure 3.6 Phylogenetic tree of Diplodia species resulting from a maximum likelihood (ML) analysis of combined ITS and tef1 datasets. Maximum likelihood bootstrap support values (BS ≥ 50 %) and Bayesian posterior probabilities (PP ≥ 0.85) are shown at the nodes (BS/PP). Ex-type strains are indicated in bold font and strains identified in this study are shown in red. Names in round parentheses are synonyms. The bar represents 0.05 substitutions per site. The tree was rooted to Botryosphaeria corticis CBS 119047 and B.

dothidea CBS 115476.

Figure 3.7 Phylogenetic tree of Dothiorella species resulting from a maximum likelihood (ML) analysis of the

ITS dataset. Maximum likelihood bootstrap support values (BS ≥ 50 %) and Bayesian posterior probabilities (PP ≥ 0.85) are shown at the nodes (BS/PP). Ex-type strains are indicated in bold font and strains identified in this study are shown in red. Names in round parentheses are synonyms. The bar represents 0.05 substitutions per site. The tree was rooted to Botryosphaeria corticis CBS 119047 and B. dothidea CBS 115476.

Pathogenicity tests

Pathogenicity was evaluated every week for up to 40 days after inoculation (Tab. 3.4).

Since there were dead plants two weeks after inoculation it was not possible to compare the lesion length, therefore a disease severity scale was used.

In the first trial, Diplodia corticola was very aggressive to cork oak while D. quercivora caused only mild symptoms although statistically different from control (H = 11.60; p < 0.01).

No symptoms were observed in the plants inoculated with Dothiorella sarmentorum.

Table 3.4 Results of the pathogenicity tests 40 days after inoculation.

Host Species Strain

Plants ¹

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1st trial

Q. suber

D. corticola ^{CH 7 E E C E E E E E E E E B B B E E E A E E} D. corticola ^{CH 60 E E E E E E E E E E E E E E E E E E E E} D. quercivora ^{CH 51 E E A A B A B B A A - - - - - - - - - -} Dothiorella sarmentorum CH 57 A A A A A A A A A A A A A A A A A A A A

Control ^{- A A A A A A A A A} A A A A A A A A A A A

2nd trial

E. globulus

N. parvum ^{PE 18} A B E E B B B B E A - - - - - - - - - - N. eucalyptorum ^{PE 139 D} A A A A A D A A A - - - - - - - - - - D. corticola ^{CH 60 A} A A A A A A A A A - - - - - - - - - -

Control ^- A A A A A A A A A A - - - - - - - - - -

E. nitens

N. parvum ^{PE 18 E E E D E E E E - - - - - - - - - - - -} N. eucalyptorum ^{PE 139} B D E E B B B A - - - - - - - - - - - - D. corticola ^{CH 60} B B B B A A E A - - - - - - - - - - - -

Control ^{- A} A A A A A A A A A - - - - - - - - - -

Q. suber

N. parvum ^{PE 18} E A E E E D A E E E - - - - - - - - - - N. eucalyptorum ^{PE 139 A} A A A A C A A C A - - - - - - - - - -

Control ^- A A A A A - - - - - - - - - - - - - - -

1 A = plant without any type of symptom; B = few dry leaves; C = half of the dry leaves; D = more than half of the leaves dry and E = completely dry plant.

On the second trial, Neofusicoccum parvum was pathogenic on the three hosts tested, with Eucalyptus nitens the most susceptible species (H = 15.00; p < 0.001). Neofusicoccum eucalyptorum significantly affected E. nitens (H = 11.67; p < 0.01) but caused minor symptoms not statistically significant from the control on E. globulus (H = 2.11; p > 0.05) and on Quercus suber (H = 12.11; p > 0.05). Diplodia corticola did not cause any symptoms on Eucalyptus globulus and only caused mild symptoms on E. nitens that were not significantly different from

the control (H = 6.82; p > 0.05). None of the control plants showed any symptom. Detailed statistical analysis is presented in the Appendix I. All inoculated fungi were reisolated from symptomatic plants, thus confirming Koch's postulates.