BACKGROUND
plant microbiome communities consist of large numbers of species  that can affect plant health, nutrition, growth and tolerance to abiotic and biotic stresses  <cit> .
a major function of plant associated microbes is to control plant pathogens, especially fungi  <cit> .
this is usually mediated by direct antagonism through production of antimicrobials, competition for nutrients or space, or induction of host plant defenses  <cit> .
due to the complexity of plant microbiomes, a rapid method is necessary to screen for their beneficial activities to plants  <cit> .
obviously, screening hundreds of these microbes in whole plants is challenging  <cit> .
researchers typically test for anti-fungal activities in vitro first, then carry forward only the positive candidates to plant-based assays, excluding the bulk of the microbes that showed no anti-pathogen activity .
dual culture screens are widely used as they are high-throughput, but it is not clear if the results correlate with microbial activities in planta .
we could find few systematic comparisons of in vitro dual culture versus in planta screens for endophytes that suppress plant disease  <cit> .
the objective of this study was to evaluate the relevance of the in vitro dual culture method as a systematic and comprehensive method for screening endophytes for anti-fungal activity.
here we screened  <dig> bacterial endophytes against fungal pathogens using in planta screens first then conducted in vitro dual culture screens.
the endophytes were previously isolated from the seeds, shoots and roots of  <dig> genotypes of the corn family   <cit> .
as zea plants are large, we used a smaller genetic grass relative for in planta screens, namely creeping bentgrass , as plants could grow and develop disease symptoms in tubes efficiently.
creeping bentgrass is one of the most widely used turfgrasses on golf courses  <cit> .
endophytes were screened for in planta suppression of two fungal pathogens, sclerotinia homoeocarpa   <cit>  and rhizoctonia solani   <cit> .
sclerotinia homoeocarpa is the most economically important disease that affects creeping bentgrass  <cit>  and is also widespread within the grass family  <cit> .
rhizoctonia solani is another major fungal pathogen that affects turfgrass species including creeping bentgrass  <cit> .
r. solani affects diverse crops including potato, tomato, cucumber, green pea and rice .
RESULTS
screening for inhibition of s. homoeocarpa
in planta visual screening of  <dig> zea endophytes for antifungal activity revealed that three endophytes  controlled s. homoeocarpa in creeping bentgrass in all 3/ <dig> tubes tested  .
the antifungal strains were previously identified based on their 16s rrna sequences   <cit> .
strains 3a <dig>  3c <dig> and 5c <dig> were identified as different isolates of burkholderia gladioli, isolated from diverse zea genotypes .
interestingly, using the in vitro dual culture screen, the same endophytes plus two additions  were found to form inhibition zones of s. homoeocarpa growth on agar in all three replicates .
strains 3h <dig> and 4h <dig> were identified as bacillus subtilis and paenibacillus polymyxa, respectively   <cit> .fig.
<dig> in vitro and in planta screening of maize endophytes for antifungal activity against s. homoeocarpa.
a-f. in planta screen.
shown are tubes with creeping bentgrass treated with a. no fungal pathogen and no endophyte, b. the pathogen but no endophyte, c-e. the pathogen and successful anti-fungal endophytes, specifically c. endophyte 3a <dig>  d. endophyte 3c <dig>  and e. endophyte 5c <dig>  f. fungicide treatment .
g-h. graphs showing the results of in vitro and in planta screening for g. endophytes 1– <dig>  and h. endophytes 96– <dig> .
the left y-axis is the plant visual health score per tube , based on the majority being very healthy , very sick  or intermediate .
an asterisk indicates that the in planta anti-fungal activity was not observed in trial  <dig> .
an example pda agar screening plate showing that zea endophytes  create an inhibition zone of s. homoeocarpa growth in vitro.
ny denotes nystatin and pcz denotes propiconazole
screening for inhibition of r. solani
in planta, out of  <dig> endophytes screened, four endophytes  controlled r. solani  in all 3/ <dig> tubes tested .
using the in vitro dual culture screen, the same endophytes, plus one addition  suppressed r. solani growth in all three replicates .
the taxonomic identities of these strains are noted above.fig.
<dig> in vitro and in planta screening of maize endophytes for antifungal activity against r. solani.
a-g. in planta screen.
shown are tubes with creeping bentgrass treated with a. no fungal pathogen and no endophyte, b. the pathogen but no endophyte, c-f. the pathogen and successful anti-fungal endophytes, specifically c. endophyte 3a <dig>  d. endophyte 3c <dig>  e. endophyte 4h <dig>  and f. endophyte 5c <dig>  g. fungicide treatment .
h-i.
graphs showing the results of in vitro and in planta screening for h. endophytes 1– <dig>  and i. endophytes 96– <dig> .
the left y-axis is the plant visual health score per tube , based on the majority being very healthy , very sick  or intermediate .
an asterisk indicates that the in planta anti-fungal activity was not observed in trial  <dig> .
the right y-axis indicates the mean diameter of the zone of inhibition of r. solani on agar plates .
j. an example pda agar screening plate showing that zea endophytes  create an inhibition zone of r. solani growth in vitro.
ny denotes nystatin and pcz denotes propiconazole
discussion
parallel in vitro and in planta screens for anti-fungal activity have previously been reported, but with limited sample sizes.
for example, amongst the largest parallel screens we could find, aravind et al.
<cit>  screened  <dig> endophytes from black pepper for activity against phytophthora capsici.
the authors identified  <dig> and 16– <dig> anti-fungal candidates based on in vitro and multiple in planta assays, respectively, but data was only presented for  <dig> strains that correlated  <cit> .
our study expands these results to  <dig> endophytes and two additional pathogens.
here, all three out of  <dig> endophytes that showed consistent anti-fungal activity against s. homoeocarpa in planta, were captured in vitro .
similarly, all  <dig> endophytes that showed consistent activity against r. solani in planta were captured in vitro .
however, the in vitro screen identified additional endophytes with anti-fungal activities that did not show activity in our in planta screen.
taking into account all  <dig> endophytes initially tested, the results from in vitro and in planta screening of endophytes that combat s. homoeocarpa positively correlated  .
for the two anti-r. solani screens, the correlation was even greater  .
excluding the in planta results that could not be replicated , the correlations were stronger  .fig.
<dig> a cartoon illustrating the correlation between the in vitro and in planta screens for zea endophytes that showed antifungal activity against both s. homoeocarpa and r. solani
our study shows that the in vitro screens captured all of the endophytes that showed anti-fungal activities in planta.
these results may be interpreted in the context of the mechanisms by which endophytes control plant pathogens: induction of host resistance, direct antagonism, or competition  <cit> .
the in vitro screen had the capacity to detect endophytes that combat pathogens mainly through direct antagonism and possibly through competition for agar nutrients, but the screen could not have captured endophytes that induce host resistance or compete for ecological plant niches .
therefore it is surprising that the in vitro assays captured all the candidates identified from the in planta screens.
there may be at least two hypotheses to explain these results:
first, the above defense mechanisms are not mutually exclusive.
it was previously reported that some antifungal compounds can also stimulate host resistance  <cit> .
hence it may be that induction of host resistance is taking place in some of the anti-fungal candidates but it is coupled with direct antagonism, allowing the endophyte  to be identified by in vitro screening.
second, the endophytes here were screened in a host different from their native host which may have affected their potential to induce host resistance or to compete with the fungal pathogen for space.
consistent with this hypothesis, in the above study by aravind et al., the authors used the native endophyte host to screen for antifungal activity and reported that at least 3/ <dig> and 2/ <dig> anti-fungal endophytes identified using two in planta screens were not apparently identified in vitro  <cit> .
here we may have been able to capture additional endophytes with anti-fungal activity in planta by testing other plant species susceptible to the same pathogens.
however, in another in planta screen using annual ryegrass  with endophytes #1- <dig> , we have observed that the same two endophytes  suppress s. homoeocarpa  as those that suppressed the pathogen in creeping bentgrass; these grasses belong to different genera.
the third anti-fungal endophyte  also suppressed s. homoeocarpa in annual ryegrass .
in the future, it would be ideal to conduct parallel screens using the  <dig> native zea hosts of these endophytes.
our in vitro screens identified “false positives” with respect to the results in planta.
this is consistent with previous studies.
for example, faltin et al.
found that out of  <dig> plant associated microbes  that inhibited r. solani in vitro,  <dig> or  <dig> microbes, respectively, inhibited r. solani in lettuce leaf discs and in sugar beets  <cit> .
in another study, 16/ <dig> phylloplane and rhizosphere bacteria that inhibited phytophthora infestans in vitro reduced disease symptoms by >10 % in planta in detached potato leaves with only two of these suppressing disease symptoms by >75 %  <cit> .
failure of some bacterial candidates from in vitro screens to suppress fungal disease when tested in planta may be attributed to many reasons, including: failure of endophytes to colonize the plant; failure of endophytes to migrate towards the pathogen; failure to compete with the native microbiota of their hosts; and/or sub-optimal production of anti-fungal compounds in planta due to pathways that compete for shared metabolic precursors, compared to optimized in vitro agar  <cit> .
alternatively, as our in planta screen was visual, it may have missed endophytes that had only weak anti-fungal activities.
CONCLUSIONS
there are practical and biological advantages and disadvantages to both in vitro and in planta screens for microbes with anti-fungal activity  <cit> .
nevertheless, our results and previous reports suggest that in vitro dual culture screening is a good method for high-throughput characterization of endophyte communities for anti-fungal activity, generally capturing the microbes that display these activities in planta.

 plant microbiome communities consist of large numbers of species that can affect plant health, nutrition, growth and tolerance to abiotic and biotic stresses. a major function of plant associated microbes is to control plant pathogens, especially fungi. this is usually mediated by direct antagonism through production of antimicrobials, competition for nutrients or space, or induction of host plant defenses. due to the complexity of plant microbiomes, a rapid method is necessary to screen for their beneficial activities to plants. obviously, screening hundreds of these microbes in whole plants is challenging. researchers typically test for anti-fungal activities in vitro first, then carry forward only the positive candidates to plant-based assays, excluding the bulk of the microbes that showed no anti-pathogen activity. dual culture screens are widely used as they are high-throughput, but it is not clear if the results correlate with microbial activities in planta. we could find few systematic comparisons of in vitro dual culture versus in planta screens for endophytes that suppress plant disease. the objective of this study was to evaluate the relevance of the in vitro dual culture method as a systematic and comprehensive method for screening endophytes for anti-fungal activity. here we screened bacterial endophytes against fungal pathogens using in planta screens first then conducted in vitro dual culture screens. the endophytes were previously isolated from the seeds, shoots and roots of genotypes of the corn family. as zea plants are large, we used a smaller genetic grass relative for in planta screens, namely creeping bentgrass, as plants could grow and develop disease symptoms in tubes efficiently. creeping bentgrass is one of the most widely used turfgrasses on golf courses. endophytes were screened for in planta suppression of two fungal pathogens, sclerotinia homoeocarpa and rhizoctonia solani. sclerotinia homoeocarpa is the most economically important disease that affects creeping bentgrass and is also widespread within the grass family. rhizoctonia solani is another major fungal pathogen that affects turfgrass species including creeping bentgrass. r. solani affects diverse crops including potato, tomato, cucumber, green pea and rice.  screening for inhibition of s. homoeocarpa in planta visual screening of zea endophytes for antifungal activity revealed that three endophytes controlled s. homoeocarpa in creeping bentgrass in all 3/ tubes tested. the antifungal strains were previously identified based on their 16s rrna sequences. strains 3a 3c and 5c were identified as different isolates of burkholderia gladioli, isolated from diverse zea genotypes. interestingly, using the in vitro dual culture screen, the same endophytes plus two additions were found to form inhibition zones of s. homoeocarpa growth on agar in all three replicates. strains 3h and 4h were identified as bacillus subtilis and paenibacillus polymyxa, respectively.fig. in vitro and in planta screening of maize endophytes for antifungal activity against s. homoeocarpa. a-f. in planta screen. shown are tubes with creeping bentgrass treated with a. no fungal pathogen and no endophyte, b. the pathogen but no endophyte, c-e. the pathogen and successful anti-fungal endophytes, specifically c. endophyte 3a d. endophyte 3c and e. endophyte 5c f. fungicide treatment. g-h. graphs showing the results of in vitro and in planta screening for g. endophytes 1– and h. endophytes 96–. the left y-axis is the plant visual health score per tube, based on the majority being very healthy, very sick or intermediate. an asterisk indicates that the in planta anti-fungal activity was not observed in trial. an example pda agar screening plate showing that zea endophytes create an inhibition zone of s. homoeocarpa growth in vitro. ny denotes nystatin and pcz denotes propiconazole screening for inhibition of r. solani in planta, out of endophytes screened, four endophytes controlled r. solani in all 3/ tubes tested. using the in vitro dual culture screen, the same endophytes, plus one addition suppressed r. solani growth in all three replicates. the taxonomic identities of these strains are noted above.fig. in vitro and in planta screening of maize endophytes for antifungal activity against r. solani. a-g. in planta screen. shown are tubes with creeping bentgrass treated with a. no fungal pathogen and no endophyte, b. the pathogen but no endophyte, c-f. the pathogen and successful anti-fungal endophytes, specifically c. endophyte 3a d. endophyte 3c e. endophyte 4h and f. endophyte 5c g. fungicide treatment. h-i. graphs showing the results of in vitro and in planta screening for h. endophytes 1– and i. endophytes 96–. the left y-axis is the plant visual health score per tube, based on the majority being very healthy, very sick or intermediate. an asterisk indicates that the in planta anti-fungal activity was not observed in trial. the right y-axis indicates the mean diameter of the zone of inhibition of r. solani on agar plates. j. an example pda agar screening plate showing that zea endophytes create an inhibition zone of r. solani growth in vitro. ny denotes nystatin and pcz denotes propiconazole discussion parallel in vitro and in planta screens for anti-fungal activity have previously been reported, but with limited sample sizes. for example, amongst the largest parallel screens we could find, aravind et al. screened endophytes from black pepper for activity against phytophthora capsici. the authors identified and 16– anti-fungal candidates based on in vitro and multiple in planta assays, respectively, but data was only presented for strains that correlated. our study expands these results to endophytes and two additional pathogens. here, all three out of endophytes that showed consistent anti-fungal activity against s. homoeocarpa in planta, were captured in vitro. similarly, all endophytes that showed consistent activity against r. solani in planta were captured in vitro. however, the in vitro screen identified additional endophytes with anti-fungal activities that did not show activity in our in planta screen. taking into account all endophytes initially tested, the results from in vitro and in planta screening of endophytes that combat s. homoeocarpa positively correlated. for the two anti-r. solani screens, the correlation was even greater. excluding the in planta results that could not be replicated, the correlations were stronger.fig. a cartoon illustrating the correlation between the in vitro and in planta screens for zea endophytes that showed antifungal activity against both s. homoeocarpa and r. solani our study shows that the in vitro screens captured all of the endophytes that showed anti-fungal activities in planta. these results may be interpreted in the context of the mechanisms by which endophytes control plant pathogens: induction of host resistance, direct antagonism, or competition. the in vitro screen had the capacity to detect endophytes that combat pathogens mainly through direct antagonism and possibly through competition for agar nutrients, but the screen could not have captured endophytes that induce host resistance or compete for ecological plant niches. therefore it is surprising that the in vitro assays captured all the candidates identified from the in planta screens. there may be at least two hypotheses to explain these results: first, the above defense mechanisms are not mutually exclusive. it was previously reported that some antifungal compounds can also stimulate host resistance. hence it may be that induction of host resistance is taking place in some of the anti-fungal candidates but it is coupled with direct antagonism, allowing the endophyte to be identified by in vitro screening. second, the endophytes here were screened in a host different from their native host which may have affected their potential to induce host resistance or to compete with the fungal pathogen for space. consistent with this hypothesis, in the above study by aravind et al., the authors used the native endophyte host to screen for antifungal activity and reported that at least 3/ and 2/ anti-fungal endophytes identified using two in planta screens were not apparently identified in vitro. here we may have been able to capture additional endophytes with anti-fungal activity in planta by testing other plant species susceptible to the same pathogens. however, in another in planta screen using annual ryegrass with endophytes #1-, we have observed that the same two endophytes suppress s. homoeocarpa as those that suppressed the pathogen in creeping bentgrass; these grasses belong to different genera. the third anti-fungal endophyte also suppressed s. homoeocarpa in annual ryegrass. in the future, it would be ideal to conduct parallel screens using the native zea hosts of these endophytes. our in vitro screens identified “false positives” with respect to the results in planta. this is consistent with previous studies. for example, faltin et al. found that out of plant associated microbes that inhibited r. solani in vitro, or microbes, respectively, inhibited r. solani in lettuce leaf discs and in sugar beets. in another study, 16/ phylloplane and rhizosphere bacteria that inhibited phytophthora infestans in vitro reduced disease symptoms by >10 % in planta in detached potato leaves with only two of these suppressing disease symptoms by >75 %. failure of some bacterial candidates from in vitro screens to suppress fungal disease when tested in planta may be attributed to many reasons, including: failure of endophytes to colonize the plant; failure of endophytes to migrate towards the pathogen; failure to compete with the native microbiota of their hosts; and/or sub-optimal production of anti-fungal compounds in planta due to pathways that compete for shared metabolic precursors, compared to optimized in vitro agar. alternatively, as our in planta screen was visual, it may have missed endophytes that had only weak anti-fungal activities.  there are practical and biological advantages and disadvantages to both in vitro and in planta screens for microbes with anti-fungal activity. nevertheless, our results and previous reports suggest that in vitro dual culture screening is a good method for high-throughput characterization of endophyte communities for anti-fungal activity, generally capturing the microbes that display these activities in planta. 
typically, plant associated microbes are screened for anti-fungal activities in vitro using the high-throughput dual culture screen, but it is not clear whether this method correlates with the activities of these microbes in planta. the objective of this study was to evaluate the relevance of the in vitro dual culture method for screening endophytes with anti-fungal activity. all endophytes that showed anti-fungal activity in planta against sclerotinia homoeocarpa and rhizoctonia solani, were captured in vitro. evidence was gained here that the in vitro dual culture method is a relevant method for high throughput screening of plant endophyte communities for anti-fungal activity. an important function of some endophytes is to suppress fungal pathogens. in parallel, bacterial endophytes from the corn grass family were screened for suppression of two fungal pathogens using the in vitro dual culture method, and in planta using the model plant, creeping bentgrass. the in vitro and in planta screening results strongly correlated. endophyteplantzeabiological controldual culturein plantacreeping bentgrasssclerotinia homoeocarparhizoctonia solaniagricultural adaptation council and ontario turfgrass research foundation052188raizada manish n. issue-copyright-statement© the author 2016 plant microbial communities consist of large numbers of endophyte species.
 endophytes are microbes that inhabit internal plant tissues without causing disease. plant microbial communities consist of large numbers of endophyte species. understanding the functions of these endophytes is a major challenge. an important function of some endophytes is to suppress fungal pathogens. typically, plant associated microbes are screened for anti-fungal activities in vitro using the high-throughput dual culture screen, but it is not clear whether this method correlates with the activities of these microbes in planta. furthermore, it is not clear whether in vitro screening captures all of the microbes that show this activity inside plants. the objective of this study was to evaluate the relevance of the in vitro dual culture method for screening endophytes with anti-fungal activity.  in parallel, bacterial endophytes from the corn grass family were screened for suppression of two fungal pathogens using the in vitro dual culture method, and in planta using the model plant, creeping bentgrass. all endophytes that showed anti-fungal activity in planta against sclerotinia homoeocarpa and rhizoctonia solani, were captured in vitro. the in vitro and in planta screening results strongly correlated.  evidence was gained here that the in vitro dual culture method is a relevant method for high throughput screening of plant endophyte communities for anti-fungal activity. in our study, the method captured all of the microbes that suppressed the corresponding pathogens in planta. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords endophyteplantzeabiological controldual culturein plantacreeping bentgrasssclerotinia homoeocarparhizoctonia solaniagricultural adaptation council and ontario turfgrass research foundation052188raizada manish n. issue-copyright-statement© the author 2016 