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. 2018 Apr 1;94(4):fiy037.
doi: 10.1093/femsec/fiy037.

Broad-spectrum inhibition of Phytophthora infestans by fungal endophytes

Sophie de Vries  1   2   3 , Janina K von Dahlen  1 , Anika Schnake  1 , Sarah Ginschel  1 , Barbara Schulz  4 , Laura E Rose  1   2   5
Affiliations

Broad-spectrum inhibition of Phytophthora infestans by fungal endophytes

Sophie de Vries et al. FEMS Microbiol Ecol. .

Abstract

Phytophthora infestans is a devastating pathogen of tomato and potato. It readily overcomes resistance genes and applied agrochemicals and hence even today causes large yield losses. Fungal endophytes provide a largely unexplored avenue of control of Phy. infestans. Not only do endophytes produce a wide array of bioactive metabolites, they may also directly compete with and defeat pathogens in planta. Here, we tested 12 fungal endophytes isolated from different plant species in vitro for their production of metabolites with anti- Phytophthora activity. Four well-performing isolates were evaluated for their ability to suppress nine isolates of Phy. infestans on agar medium and in planta. Two endophytes reliably inhibited all Phy. infestans isolates on agar medium, of which Phoma eupatorii isolate 8082 was the most promising. It nearly abolished infection by Phy. infestans in planta. Our data indicate a role for the production of anti-Phytophthora compounds by the fungus and/or an enhanced plant defense response, as evident by an enhanced anthocyanin production. Here, we present a potential biocontrol agent, which can inhibit a broad-spectrum of Phy. infestans isolates. Such broadly acting inhibition is ideal, because it allows for effective control of genetically diverse isolates and may slow the adaptation of Phy. infestans.

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Figures

Figure 1.
Figure 1.
Phylogenetic placement of fungal endophytes. Neighbor-Joining phylogeny of ascomycetes closely related to the four fungal endophytes (A-D). Cloned sequences are shown in purple and the best BLAST hit is shown in blue. The monophyletic clade of the genus Monosporascus is indicated by the blue dot (D). The trees are rooted with Leotia lubrica (A), Ascochyta hordei var. hordei (B), Dothidea hippophaeos (C) and Valsaria ceratoniae (D). Only bootstrap values >50 are shown. The bar below the phylogeny indicates the distance measure for the branches. (E) Pictures of the four fungal endophytes on plates, as well as close-ups of the mycelial growth on plates, microscopic pictures of hyphal growth in culture and in roots of S. lycopersicum. The cultures of Pho. eupatorii show pycnidia (black arrows) as well as chlamydospores (purple arrow) and pycnidiospores (grey arrow). Note the primarily intercellular growth of Pho. eupatrii in planta (black arrows), which may contribute to its asymptomatic root colonization of S. lycopersicum. Phi. fortinii, in contrast, colonized both inter- and intracellularly, as was observed for this isolate in other hosts (black arrow). Scale bars are given in the corner of each photograph.
Figure 2.
Figure 2.
Co-cultivation of fungal endophytes with Phytophthora infestans on agar culture medium. Examples of two-week-old single and co-cultures of Phialocephala fortinii with Phy. infestans isolate 3928A (A,E,I), Phoma eupatorii with Phy. infestans isolate NL90128 (B,F,J) and 9907 with Phy. infestans isolate T15-2 (C,G,K) and eight-day old single and co-cultures of Monosporascus sp. with Phy. infestans isolate D12-2 (D,H,L). The diameter of each Petri dish is 9 cm. Radial growth inhibition of Phy. infestans isolates by fungal endophtyes (M-P). Radial growth (R) of the different Phy. infestans isolates denoted on the x-axis when grown alone (orange) or in co-culture with the four fungal endophytes (blue): Phi. fortinii (M), Pho. eupatorii (N), isolate 9907 (O) and Monosporascus sp. (P). At least 10 biological replicates per control or co-cultivation were measured. The box indicates the upper and lower 50% quartile (interquartile range, IQR), the horizontal line in each box shows the median, the whiskers indicate the upper and lower bounds of the 1.5x IQR and the circles show data points, which are outliers. Significant differences are noted as *P < 0.05, **P < 0.01, ***P < 0.001 and ns = not significant.
Figure 3.
Figure 3.
Long-term co-cultivation of fungal endophytes with Phytophthora infestans on agar growth medium. Examples of eight-week-old co-cultures and their respective controls. Phi. fortinii with Phy. infestans isolate NL88069 (A,E,I), Pho. eupatorii with Phy. infestans isolate NL88069 (B,F,J), isolate 9907 with Phy. infestans isolate T15-2 (C,G,K) and Monosporascus sp. with Phy. infestans isolate NL10001 (D,H,L). The diameter of each Petri dish is 9 cm.
Figure 4.
Figure 4.
In planta co-inoculations of Phoma eupatorii isolate 8082 and Phytophthora infestans. Solanum lycopersicum cv. M82 seedlings were mock treated (A) or inoculated with Phy. infestans isolate D12-2 (B), 5 µl of Pho. eupatorii mycelium suspension (C), 10 µl of Pho. eupatorii mycelium suspension (D), 5 µl of Pho. eupatorii mycelium suspension and Phy. infestans isolate D12-2 (E) and 10 µl of Pho. eupatorii mycelium suspension and Phy. infestans isolate D12-2 (F). Chlorophyll fluorescence is depicted in red false coloring for all combinations (G-L) and was measured as mean fluorescence intensity using ImageJ2 (M). Bars give the average mean fluorescence (nleaflets = 17–37). Error bars give the standard error of the mean (SEM); ns = not significant. Differences in anthocyanin content (N). A darker pink in the examples shown indicates a higher concentration of anthocyanins in the sample. Average relative anthocyanin content with standard deviation is given in brackets following each treatment. In total, three to six biological replicates per treatment were analyzed. The average relative necrotic area of the leaflets was calculated for each treatment (nleaflets = 38–156). Bars give the average necrotic area per treatment and error bars indicate the SEM. Significant differences between the treatments are indicated by different letters above the bars with a cutoff of P < 0.05; same letter = not significant. The relative abundance of Phy. infestans isolate D12-2 was measured with a qRT-PCR of the two biomass marker genes PiH2a and PiElf1α (P). Bars show average relative expression of the two biomass markers normalized against the three plant reference genes SAND, TIP and TIF3H. It compares relative abundance of Phy. infestans in Pho. eupatorii–Phy. infestans co-inoculations with that in control treatments (Phy. infestans only). Three biological replicates per treatment were used in all cases except for Pho. eupatorii (10 µl mycelial suspension) with Phy. infestans, in which only two biological replicates were used. The error bars indicate the SEM. Significant differences between relative Phy. infestans abundance in samples pre-inoculated with the endophyte and the control are indicated by *P < 0.05 and **P < 0.01. In all bar graphs, treatments with Pho. eupatorii are indicated by its isolate number 8082.
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