Growth, Survival and Spore Formation of the Pathogenic Aquatic Oomycete Aphanomyces astaci and Fungus Fusarium avenaceum Are Inhibited by Zanthoxylum rhoifolium Bark Extracts In Vitro

: This study aimed to evaluate the in vitro activity of Zanthoxylum rhoifolium bark (Zr-b) extracts against pathogenic aquatic oomycete/fungal isolates that cause different diseases in native European crayﬁsh resulting in an elevated mortality rate and severe economic repercussions. n -hexane, chloroform, chloroform–methanol (9:1) and methanol extracts of Zr-b were used to evaluate the antifungal activity against the strain UEF88662 of Aphanomyces astaci (oomycete) and the strain SMM2 of Fusarium avenaceum (fungus). The anti-oomycete and antifungal activity was quantitatively evaluated by growth, survival and sporulation microbiological assays. The extracts tested demonstrated a dose-dependent inhibitory effect on oomycete and fungal growth and survival, as well as on the production of oomycete and fungal spores. This work presents alternatives for the treatment and prevention of the spreading of Aphanomyces astaci and Fusarium avenaceum , the etiological agents of the diseases crayﬁsh plague and brown spot disease, respectively. The antifungal properties of Zanthoxylum rhoifolium bark extracts warrant further research on their use in the prevention and treatment of both oomycete and fungal diseases. The antifungal properties of Zanthoxylum rhoifolium bark extracts, shown in vitro, indicate the possibility of their use in new therapeutic and prophylactic strategies, providing perspectives for the design of in vivo studies.


Introduction
Fungal diseases are often caused by species that are common in the environment. Although most fungi are not dangerous, some types can be harmful to health [1]. Fusarium avenaceum belongs to the phylum Ascomycota, class Sordariomycetes, order Hypocreales, family Nectreaceae. It is found worldwide and commonly isolated from soil and many different species of plants [2]. Many important plant pathogens and mycotoxin producers, including saprophytes and endophytes, belong to the genus extracts showed a fungistatic effect on different fungal plant pathogens, such as, Botrytis cinerea, Sclerotinia sclerotiorum, Alternaria alternata, Colletotrichum gloeosporioides and Clonostachys rosea [36].
In this context, we studied the effects of extracts of the bark of Zanthoxylum rhoifolium on the growth, survival and spore formation of an oomycete and a fungus that cause severe diseases in crayfish: Aphanomyces astaci and Fusarium avenaceum.

In Vitro Anti Oomycete and Antifungal Activity of Zr-b Extracts
The antioomycete and antifungal activity of all the Zr-b extracts was assayed against crayfish isolates of Aphanomyces astaci (UEF88662 strain) and Fusarium avenaceum (SMM2 strain), respectively ( Figure 1). extracts showed a fungistatic effect on different fungal plant pathogens, such as, Botrytis cinerea, Sclerotinia sclerotiorum, Alternaria alternata, Colletotrichum gloeosporioides and Clonostachys rosea [36].
In this context, we studied the effects of extracts of the bark of Zanthoxylum rhoifolium on the growth, survival and spore formation of an oomycete and a fungus that cause severe diseases in crayfish: Aphanomyces astaci and Fusarium avenaceum.

In Vitro Anti Oomycete and Antifungal Activity of Zr-b Extracts
The antioomycete and antifungal activity of all the Zr-b extracts was assayed against crayfish isolates of Aphanomyces astaci (UEF88662 strain) and Fusarium avenaceum (SMM2 strain), respectively ( Figure 1).

Figure 1.
In the upper panel, Aphanomyces astaci strain 88662 (A) and Fusarium avenaceum strain SMM2 (B) grown on Potato-Dextrose (PD) agar (CONDA), a selective medium for the growth of yeasts and molds. In the lower panel, the injuries on the exoskeleton of a crayfish caused by Aphanomyces astaci, the etiological agent of "crayfish plague" (C) and Fusarium avenaceum, the etiological agent of the "burn spot disease" (D).
The diameters of the inhibition zones, obtained with 6 mg/disc of the Zr-b extracts against the two isolates, are reported in Table 1 and Figure 2. In the lower panel, the injuries on the exoskeleton of a crayfish caused by Aphanomyces astaci, the etiological agent of "crayfish plague" (C) and Fusarium avenaceum, the etiological agent of the "burn spot disease" (D).
The diameters of the inhibition zones, obtained with 6 mg/disc of the Zr-b extracts against the two isolates, are reported in Table 1 and Figure 2.  The inhibition zone (mm) is reported as mean ± standard deviation (SD). The extraction buffers were used as negative controls and showed no effects on tested microorganisms. The results of in vitro assays indicate that all the Zr-b extracts exhibited a clear antioomycete and antifungal activity, with the formation of wide inhibition zones against Fusarium avenaceum and Aphanomyces astaci, although the widest area of inhibition was obtained with the chloroform-methanol extract. Tioconazole (TCZ) (1.4 mg/disc), used as a positive control, exerted the expected antifungal activity against Fusarium avenaceum with an inhibition zone of 40.1 ± 7.2 mm, but also induced an inhibition zone of 20.0 ± 4.1 mm against Aphanomyces astaci. The extraction buffers, used as negative controls, showed no effects toward the tested microorganisms. The results indicate that the SMM2 Fusarium avenaceum strain was more sensitive to all the Zr-b extracts tested and to Tioconazole, compared to the UEF88662 Aphanomyces astaci strain. This result was also confirmed by quantitative assays.

Effect of Zr-b Extracts on Oomycete and Fungal Growth and Survival
The antioomycete and antifungal activities of all the Zr-b extracts have been confirmed by quantitative assays. All Zr-b extracts showed both oomycestatic and fungistatic effects other than oomyceticidal and fungicidal effects ( Table 2). The growth of UEF88662 Aphanomyces astaci isolate was inhibited by n-hexane, chloroform, chloroform-methanol (9:1) and methanol Zr-b extracts at a concentration of 40 μg/μL. The growth of SMM2 Fusarium avenaceum isolate was inhibited by lower concentrations of n-hexane, chloroform, chloroform-methanol (9:1) and methanol Zr-b extracts equal to 5 μg/μL. The minimum inhibitory concentration (MIC) values for the TCZ were 1.5 μg/μL and 1.0 μg/μL for Aphanomyces astaci and Fusarium avenaceum, respectively.  The results of in vitro assays indicate that all the Zr-b extracts exhibited a clear antioomycete and antifungal activity, with the formation of wide inhibition zones against Fusarium avenaceum and Aphanomyces astaci, although the widest area of inhibition was obtained with the chloroform-methanol extract. Tioconazole (TCZ) (1.4 mg/disc), used as a positive control, exerted the expected antifungal activity against Fusarium avenaceum with an inhibition zone of 40.1 ± 7.2 mm, but also induced an inhibition zone of 20.0 ± 4.1 mm against Aphanomyces astaci. The extraction buffers, used as negative controls, showed no effects toward the tested microorganisms. The results indicate that the SMM2 Fusarium avenaceum strain was more sensitive to all the Zr-b extracts tested and to Tioconazole, compared to the UEF88662 Aphanomyces astaci strain. This result was also confirmed by quantitative assays.

Effect of Zr-b Extracts on Oomycete and Fungal Growth and Survival
The antioomycete and antifungal activities of all the Zr-b extracts have been confirmed by quantitative assays. All Zr-b extracts showed both oomycestatic and fungistatic effects other than oomyceticidal and fungicidal effects ( Table 2). The growth of UEF88662 Aphanomyces astaci isolate was inhibited by n-hexane, chloroform, chloroform-methanol (9:1) and methanol Zr-b extracts at a concentration of 40 µg/µL. The growth of SMM2 Fusarium avenaceum isolate was inhibited by lower concentrations of n-hexane, chloroform, chloroform-methanol (9:1) and methanol Zr-b extracts equal to 5 µg/µL. The minimum inhibitory concentration (MIC) values for the TCZ were 1.5 µg/µL and 1.0 µg/µL for Aphanomyces astaci and Fusarium avenaceum, respectively. Furthermore, the minimum oomycetidal concentration (MOC) of n-hexane, chloroform, chloroform-methanol (9:1) and methanol Zr-b extracts was 80 µg/µL for UEF88662 Aphanomyces astaci strain.
The minimum fungicidal concentration (MFC) of n-hexane, chloroform, chloroform-methanol (9:1) and methanol Zr-b extracts was 40 µg/µL for SMM2 Fusarium avenaceum strain. Both the oomycete and fungal isolates used in this study were sensitive to TCZ, with a MOC value of 15 µg/µL for Aphanomyces astaci and MFC value of 1.5 µg/µL for Fusarium avenaceum.

Effect of the Chloroform-Methanol (9:1) Zr-b Extract on Oomycete and Fungal Fitness
The fitness of the pathogenic aquatic oomycete and fungal isolates was monitored in the presence of different concentrations (0-80 µg/µL) of the chloroform-methanol (9:1) Zr-b extract ( Figure 3A,B); however, the growth curves were not reported because the spectrophotometric determination was distorted by the turbidity of the extracts. The figures show that the extract interferes with both the oomycete and fungal survival of UEF88662 Aphanomyces astaci strain and SMM2 Fusarium avenaceum strain in a dose-dependent manner. Indeed, the number of viable cells was reduced proportionally with the increasing concentration of the extract during the 96 h of observation. Fusarium avenaceum was more sensitive to the chloroform-methanol (9:1) Zr-b extract compared to Aphanomyces astaci. Furthermore, the minimum oomycetidal concentration (MOC) of n-hexane, chloroform, chloroform-methanol (9:1) and methanol Zr-b extracts was 80 μg/μL for UEF88662 Aphanomyces astaci strain. The minimum fungicidal concentration (MFC) of n-hexane, chloroform, chloroformmethanol (9:1) and methanol Zr-b extracts was 40 μg/μL for SMM2 Fusarium avenaceum strain. Both the oomycete and fungal isolates used in this study were sensitive to TCZ, with a MOC value of 15 μg/μL for Aphanomyces astaci and MFC value of 1.5 μg/μL for Fusarium avenaceum.

Effect of the Chloroform-Methanol (9:1) Zr-b Extract on Oomycete and Fungal Fitness
The fitness of the pathogenic aquatic oomycete and fungal isolates was monitored in the presence of different concentrations (0-80 μg/μL) of the chloroform-methanol (9:1) Zr-b extract ( Figure 3A,B); however, the growth curves were not reported because the spectrophotometric determination was distorted by the turbidity of the extracts. The figures show that the extract interferes with both the oomycete and fungal survival of UEF88662 Aphanomyces astaci strain and SMM2 Fusarium avenaceum strain in a dose-dependent manner. Indeed, the number of viable cells was reduced proportionally with the increasing concentration of the extract during the 96 h of observation. Fusarium avenaceum was more sensitive to the chloroform-methanol (9:1) Zr-b extract compared to Aphanomyces astaci. The experiments were performed in triplicate and statistical significance was examined by the twoway ANOVA test with a Bonferroni correction. Results are indicated as means ± standard deviations (SDs). Asterisks indicate statistical significance (* p < 0.05; ** p < 0.001; *** p < 0.0001).

Effect of the Chloroform-Methanol (9:1) Zr-b Extract on Oomycete and Fungal Sporulation and Spore Germination
After the evaluation and quantification of the inhibitory effect of the chloroform-methanol (9:1) Zr-b extract on the vegetative form of UEF88662 Aphanomyces astaci strain and SMM2 Fusarium avenaceum strain, we evaluated the ability of the chloroform-methanol extract to inhibit the production of spores from both strains. The sporulation inhibition assay show that the inhibitory effect of Zr-b extracts on both oomycete and fungal spore production was dose-dependent, with complete inhibition of the sporulation at a concentration of 320 μg/μL ( Figure 4A,B).  After the evaluation and quantification of the inhibitory effect of the chloroform-methanol (9:1) Zr-b extract on the vegetative form of UEF88662 Aphanomyces astaci strain and SMM2 Fusarium avenaceum strain, we evaluated the ability of the chloroform-methanol extract to inhibit the production of spores from both strains. The sporulation inhibition assay show that the inhibitory effect of Zr-b extracts on both oomycete and fungal spore production was dose-dependent, with complete inhibition of the sporulation at a concentration of 320 µg/µL ( Figure 4A,B). To verify the sporicidal effect of the Zr-b extract, a spore germination inhibition assay was performed in vitro with 320 μg/μL chloroform-methanol (9:1) Zr-b extract. After the appropriate incubation, no development of viable colonies of Aphanomyces astaci and Fusarium avenaceum was observed on media supplemented with the Zr-b extract ( Figure 5)  To verify the sporicidal effect of the Zr-b extract, a spore germination inhibition assay was performed in vitro with 320 µg/µL chloroform-methanol (9:1) Zr-b extract. After the appropriate incubation, no development of viable colonies of Aphanomyces astaci and Fusarium avenaceum was observed on media supplemented with the Zr-b extract ( Figure 5) To verify the sporicidal effect of the Zr-b extract, a spore germination inhibition assay was performed in vitro with 320 μg/μL chloroform-methanol (9:1) Zr-b extract. After the appropriate incubation, no development of viable colonies of Aphanomyces astaci and Fusarium avenaceum was observed on media supplemented with the Zr-b extract ( Figure 5)

Discussion
The results obtained from the present study show that the Zanthoxylum rhoifolium bark extracts possess both oomycetidal and antifungal activity and in vitro can antagonize the growth of both Aphanomyces astaci and Fusarium avenaceum isolates. Zanthoxylum has been studied for several types of biological actions including the antifungal activity [42].
All Zr-b extracts were effective against Aphanomyces astaci and Fusarium avenaceum, counteracting their growth in the agar-diffusion assay; the chloroform-methanol (9:1) extract in particular exhibited a wider inhibition zone than n-hexane, chloroform and methanol extracts, suggesting that the efficacy of the organic solvents to extract the active principles is more or less equivalent. Extracts from medicinal plants have been extensively used from ancient times against fungal infections for treating various disease conditions in humans and in agriculture [43]. Ethanolic extracts of the bark of Zanthoxylum fagara, Zanthoxylum elephantiasis and Zanthoxylum martinicense showed antifungal activity [44], as well as ether, chloroform and methanol extracts of Zanthoxylum budrunga bark [45]. The Zanthoxylum armatum essential oil has been shown to inhibit the mycelial growth of the fungus Bipolaris sorokiniana [46]. Biological activity against fungi has been reported also in the bark of Zanthoxylum usambarense [47] and Zanthoxylum americanum [9]. The fungicidal activity is likely due to the presence of alkaloids, the major components, along with flavonoids, within the Zanthoxylum genus [48]. The alkaloidal extract of the bark of Zanthoxylum chiloperone exhibited antifungal activity against Candida albicans, Aspergillus fumigatus and Trichophyton mentagrophytes [49]. Moreover, a synergistic action against fluconazole-resistant Candida albicans clinical strains was observed with concomitant use of berberine and fluconazole [50]. Because of their good antifungal properties and synergistic action, berberine and its derivatives have been suggested to represent a new class of antifungal agents with low host toxicity [51]. Interestingly, we found that the most abundant bioactive molecules in the bark of Zanthoxylum rhoifolium were N,O-dimethylthaicanine and coralydine, two protoberberine-derived alkaloids. Protoberberine alkaloids display a great variety of biological and pharmacological activities. Such activities include the inhibition of DNA synthesis, protein biosynthesis, the inhibition of membrane permeability, and the uncoupling of oxidative phosphorylation. These processes likely explain the allelochemical and toxic effects observed against bacteria, fungi, other plants, insects, and vertebrates [52].
Other compounds found in the present study in Zr-b were the flavonoids 3-OMe-benzoyl-1-β-D-(6-galloyl)-glucopyranoside, hesperidin (aflavonoid glycoside) and isorhamnetin-3-0-rutinoside (an O-methylated flavonol), already reported in Zanthoxylum schinifolium by Li et al. [48]. Flavonoids have been reported to possess many biological properties including antimicrobial [53] and antifungal [54] activities. Owing to the widespread ability of flavonoids to inhibit spore germination in plant pathogens, they have been proposed to be useful against human fungal pathogens [55] and their activity against the opportunistic pathogen Candida albicans has been shown [56,57]. Galangin, a flavonol commonly found in bee propolis samples [58], has been shown to have inhibitory activity against Aspergillus tamari and Aspergillus flavus, fungi responsible for massive diseases in immunosuppressed patients [59,60].
The MIC values of the Zanthoxylum rhoifolium extracts against Aphanomyces astaci and Fusarium avenaceum isolates showed that the extracts were oomycestatic and fungistatic at lower concentrations while becoming oomycetidal and fungicidal at higher concentrations. It is worth noting that the quantitative microbiological tests highlighted the higher antioomycete and antifungal activity of the chloroform-methanol (9:1) Zr-b extract in comparison to Tioconazole, used as a positive control. Indeed, according to the HPLC quantification, the amount of alkaloids and flavonoids in the chloroform-methanol extract was 5.9 mg/g. This allows us to estimate a MIC of 0.24 and 0.003 µg/µL for Aphanomyces astaci and Fusarium avenaceum, respectively; and a MOC of 0.48 for Aphanomyces astaci and an MFC of 0.24 µg/µL for Fusarium avenaceum, concentrations well below the MIC, MOC and MFC of Tioconazole. Numerous natural substances possess both antimicrobial and antifungal activity more powerful than the pharmaceutical products of chemical synthesis [51].
We hypothesize that the reason for such high antioomycete and antifungal activities, shown by the chloroform-methanol (9:1) Zr-b extract, can reside in the synergistic effect of the different phytochemicals present in the extract. The crude extract of Spirulina spp., showed improved antifungal activity against Fusarium graminacearum, compared to the single phenolic acids. The improvement occurred also when the crude extract was mixed with the purified standard gallic acid [61]. It is interesting to note that the research has been focusing on novel natural products to substitute the chemical synthesis products that often show high toxicity and the prolonged use is accompanied by the drawback of the onset of resistant strains, that can be minimized by the use of blends of natural molecules [62].
In vitro, the chloroform-methanol (9:1) Zr-b extract is able to effectively inhibit the spore production from Aphanomyces astaci and Fusarium avenaceum isolates, with sporicidal activity to high concentrations. As expected, the minimum sporicidal concentration of Zr-b extracts is four-fold higher than the MOC for Aphanomyces astaci and eight-fold higher than the MFC for Fusarium avenaceum, being generally the mycotic spores more resistant than the vegetative forms to the effects of natural and synthetic antimycotics which often have no or very low influence on the spores [63,64].
These properties are very important as the sporulation is crucial to the potential spreading of these disease agents. Although many chemicals had antifungal properties, some of them are very toxic (such as formaldehyde, malachite green, etc.) and prohibited in aquaculture, while for others (such as hydrogen peroxide, acetic acid, povidone iodine, etc.) the effective concentrations are higher than the levels recommended by the U.S. Food and Drug Administration, and therefore they may cause adverse effects on animal health, and in general may be injurious to aquatic biota [65].
To conclude, the results of the microbiological tests performed in this study on the evaluation of the inhibitory effects of the Zr-b extracts on the survival and sporulation of Aphanomyces astaci and Fusarium avenaceum, suggest that these extracts could be used as means to prevent the spreading of the tested oomycete and fungal disease agents and encourage the promotion of their use as alternatives to chemicals in the prevention and treatment of diseases, that represent a real scourge, especially in crayfish aquaculture. Additionally, the present study constitutes a basis for further investigations on molecular mechanisms of antioomycete and antifungal activities of the Zr-b extracts and for the design of in vivo studies. The extracts of the plant used in this study could be useful in the treatment and prevention of infections caused by these two pathogens. However, further studies are required to determine the cost, applicability, and safety of these extracts as potential antioomycetes and antifungals.

Microorganisms and Growth Conditions
The strains UEF88662 of Aphanomyces astaci and SMM2 of Fusarium avenaceum (crayfish isolates) were provided by the Department of Biology Kuopio Campus, University of Eastern Finland. The microorganisms were cultured in Potato-Dextrose (PD) broth/agar (CONDA, Torrejón de Ardoz, Spain), a medium developed for cultivation of yeasts and molds, at 20 • C. Both strains were maintained at 4 • C on agar media. The isolates were stored frozen at −80 • C in PD broth supplemented with 10% glycerol (v/v) (Carlo Erba Reagents, Milan, Italy) until use and the working cultures were activated in the PD broth at 20 • C for 24-48 h.

Plant Material
The bark of Zanthoxylum rhoifolium Lam was collected in Venezuela and identified by Eng. Juan Carmona, Universidad de Los Andes, Merida, Venezuela. A voucher specimen (No. 607) was deposited to Jardin de Plantas Medicinales de la Facultad de Farmacia y Bioanalisis, Merida.

Extraction and Characterization of Zanthoxylum rhoifolium bark
An amount of 810 g of Zr-b was sequentially extracted with solvents of increasing polarity yielding n-hexane, chloroform, chloroform-methanol (9:1) and methanol extracts, in the amounts of 25.6 g, 16.08 g, 50.48 g, and 94.12 g, respectively (3.14%, 2.06%, 6.19% and 11.55% with respect to the dry plant material). The chloroform-methanol (9:1) extract was partitioned between n-butanol and H 2 O to remove the sugar portion and subjected (2.5 g) to Column Chromatography (CC) over Sephadex LH-20 in MeOH to give seven major fractions (A-G) grouped by Thin Layer Chromatography (TLC). TLC was performed on precoated Kieselgel 60 F254 plates (Merck). Only the most abundant fractions (B and E) were subjected to further purification. Fraction B (466.3 mg) was purified by reverse-phase high performance liquid chromatography on a Shimadzu LC-20AT series pumping system equipped with a Shimadzu RID10A refractive index detector and a Shimadzu injector, using a C18µ-Bondapak column (30 cm

In Vitro Antioomycetidal and Antifungal Activity Assay of Zr-b Extracts with the Agar-Diffusion Method
In order to evaluate the inhibitory spectrum of Zr-b extracts, against test microorganisms, a variation of the agar-diffusion method was performed [66]. Briefly, the oomycete and fungal strains were grown in PD broth to an optical density of 0.5 at 600 nm; an aliquot of 200 µL of oomycete and fungal suspension, appropriately homogenized to generate fragmented hyphae, was spread on the surface of agar media. Paper disks (6 mm in diameter, Oxoid, S.p.a., Rodano, Milano, Italy), impregnated with 20 µL of Zr-b extracts, were positioned on media using sterile forceps. Different concentrations of Zr-b extracts (1-6 mg/disc) were used to evaluate both antioomycetidal and antifungal activities. TCZ (Pfizer Italia Srl, Latina, Italy), at a concentration of 1.4 mg/disc was used as positive control. The extraction buffers, n-hexane, chloroform, methanol and chloroform-methanol (9:1), were used as negative controls. Plates were incubated at 20 • C for 2-5 days. The size (expressed in mm) of the inhibition zones (including disc diameter) around the disc was measured. The antioomycetidal and antifungal activities were expressed as the diameter of the inhibition zones produced by the Zr-b extracts against the test microorganisms. The experiments were repeated three times.

Quantitative Evaluation of the Antioomycetidal and Antifungal Activities of Zr-b Extracts
The susceptibility of both oomycete and fungal strains to different concentrations of Zr-b extracts was determined by the dilution tube method, with 1 × 10 5 CFU/mL as standard inoculums [67]. Zr-b extracts were added in a series of tubes achieving final concentrations of 0, 5, 10, 20, 40, 80, and 160 µg/µL, and incubated at 20 • C for 2-5 days. The isolates were also tested with TCZ as positive control and with the extraction buffer as negative control. After the incubation, an aliquot of the suspension of hyphae fragments from each tube was used to determine the optical density at 600 nm, while an aliquot was spread on the surfaces of PD agar plates in duplicate. Plates were then incubated at 20 • C for 2-5 days, and a colony count was performed. The MIC was assigned to the lowest concentration of Zr-b extract which prevented fungal growth. The MOC and the MFC were defined as the minimum extract concentration that killed 99% of oomycete and fungi respectively from the initial inoculums. The experiments were repeated three times.

Oomycete and Fungal Fitness Evaluation of the Chloroform-Methanol (9:1) Zr-b Extract
To verify the effect of Zr-b extracts on the fitness of Aphanomyces astaci and Fusarum avenaceum, the assays of oomycete and fungal growth and assays of oomycete and fungal survival were performed in the presence of increasing concentrations of chloroform-methanol (9:1) Zr-b extracts. To evaluate the growth of each strain, during the observation period (96 h), aliquots of serial dilutions of the oomycete and fungal suspensions were used to determine the optical density at 600 nm. To evaluate the survival of each strain, during the observation period (96 h), aliquots of serial dilutions of the oomycete and fungal suspensions were spread on PD agar, and the plates were incubated at 20 • C for 2-5 days. Thereafter, the count of oomycete and fungal colonies on agar was carried out. All experiments were performed in triplicate with three independent cultures. 4.7. Assays of Oomycete and Fungal Sporulation with the Chloroform-Methanol (9:1) Zr-b Extract In order to evaluate the inhibitory effect of the chloroform-methanol (9:1) Zr-b extract on the spore production of Aphanomyces astaci and Fusarium avenaceum, sporulation inhibition assays were performed in the presence of increasing concentrations of the extract. To induce the production of spores, a variation of the procedure described by Makkonen et al. was performed [68], both for Aphanomyces astaci and for Fusarium avenaceum. Briefly, the oomycete and the fungus were grown for 2-5 days on PD agar and a few millimeters of agar layer covered with mycelia was transferred in 1 mL of PD broth. After 1 week of incubation at 20 • C, a filtration was carried out with sterile gauze in order to remove the agar layer, and 1 mL of sterile distilled water was added. After 24 h of incubation at 20 • C, a count of oomycete and fungal spores was carried out under an optical microscope (Nikon Eclipse E600, NITAL Spa, Turin, Italy) with a Burker chamber. For the sporulation inhibition assays, the Zr-b extracts were added to a final concentration of 0, 5, 10, 20, 40, 80, 160, and 320 µg/µL, initially in growth medium and subsequently in sterile distilled water during the incubation of the mycelia in nutritional stress. All experiments were performed in triplicate.

Assays of Spore Germination with the Chloroform-Methanol (9:1) Zr-b Extract
To verify and confirm the efficacy on the plate of the minimum sporicidal concentration value obtained from the previous experiment, we designed an assay of zoospore and spore germination inhibition. The experiments were performed with 320 µg/µL chloroform-methanol (9:1) Zr-b extract, a concentration that completely abolished the in vitro oomycete and fungal sporulation. Briefly, 1 mL of PD broth, containing 2 × 10 4 zoospores of Aphanomyces astaci and spores of Fusarium avenaceum, was spread on PD agar extract-free (as a positive control) and on PD agar supplemented with 320 µg/µL of chloroform-methanol (9:1) Zr-b extracts. After 1 week of incubation at 20 • C, the oomycete and fungal growth was verified. The experiment was performed in triplicate.

Statistical Analysis
Data are reported as mean ± standard deviation (SD). Data were analyzed and graphically reported by using "GraphPad Prism 4" software, validating the statistical significance of the two-way ANOVA test with a Bonferroni correction. In all cases, p values lower than 0.05 were considered statistically significant.