Natural occurrence of the entomopathogenic fungi Beauveria
bassiana as a vertically transmitted endophyte of Pinus
radiata and its effect on above- and below-ground insect
pests
Marie-Caroline LEFORT, Aimee C McKinnon, Tracey L Nelson, Travis R Glare
Background. The New Zealand forest industry would greatly benefit from a successful
way of controlling insect pests. The entomopathogenic fungus, Beauveria bassiana could
hold such potential and has previously been shown to be capable of endophytic
colonisation of the Monterey pine Pinus radiata. Nevertheless clarifications on its mode of
transmission, persistence and action in this plant are required. In this study we
investigated B. bassiana transmission and persitence in P. radiata and whether this fungus
is beneficial to P. radiata by testing its effect as a plant endophyte on the fitness
performance of above and belowground insect feeders.
Methods. Both culturing and molecular approaches were used to detect the occurrence B.
bassiana in pines. Transmission electron microscopy of positive germinating seeds was
also used to locate the fungus. Bioassays were conducted on root and needle feeding
insects using Beauveria positive and endophyte free pine seedlings.
Results. Beauveria bassiana was detected in seedlings which had not previously been
exposed to the fungus, indicating a vertical mode of transmission. The fungus could
colonise all parts of the pines, but did not always persist. We found that the presence of
the fungus negatively affects the fitness of the below-ground insect feeding on the plant
by reducing their survival by over 10% and their weight by 5%. This study also showed
that the mode of action of endophytic B. bassiana in pine is likely to be by feeding
deterrence of insects induced locally by fungal metabolites, rather than by direct fungal
infection of the insects.
Discussion. A vertically transmitted beneficial endophyte of pine could be used as a cost
effective approach to control insect pest in these commercially grown trees.
PeerJ PrePrints | https://doi.org/10.7287/peerj.preprints.1632v1 | CC-BY 4.0 Open Access | rec: 6 Jan 2016, publ: 6 Jan 2016
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Natural occurrence of the entomopathogenic fungi Beauveria
bassiana as a vertically transmitted endophyte of Pinus radiata and
its effect on above- and below-ground insect pests
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Authors:
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Marie-Caroline LEFORT*, (1) Bio-Protection Research Centre, PO Box 85084, Lincoln
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University, Lincoln 7647, Christchurch, New Zealand (2) Department of Natural Sciences, Unitec
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Institute of Technology, Auckland, New Zealand
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Aimee C. McKINNON, Bio-Protection Research Centre, PO Box 85084, Lincoln University,
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Lincoln 7647, Christchurch, New Zealand.
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Tracey L. NELSON, AgResearch, PO Box 4749, Christchurch 8140, New Zealand
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Travis R. GLARE, Bio-Protection Research Centre, PO Box 85084, Lincoln University,
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Lincoln 7647, Christchurch, New Zealand.
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*Corresponding
author, Marie-Caroline.Lefort@lincolnuni.ac.nz, mlefort@unitec.ac.nz
Ph: 09 815 4321 ext. 8075
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Natural occurrence of the entomopathogenic fungi Beauveria bassiana as a vertically
transmitted endophyte of Pinus radiata and its effect on above- and below-ground insect
pests
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ABSTRACT
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Background. The New Zealand forest industry would greatly benefit from a successful way of
30
controlling insect pests. The entomopathogenic fungus, Beauveria bassiana, could hold such
31
potential and has previously been shown to be capable of endophytic colonisation of the Monterey
32
pine Pinus radiata. Nevertheless clarifications on its mode of transmission, persistence and action
33
in this plant are required. In this study we investigated B. bassiana transmission and persistence in
34
P. radiata and whether this fungus is beneficial to P. radiata by testing its effect as a plant
35
endophyte on the fitness performance of above and belowground insect feeders.
36
Methods. Both culturing and molecular approaches were used to detect the occurrence B. bassiana
37
in pines. Transmission electron microscopy of positive germinating seeds was also used to locate
38
the fungus. Bioassays were conducted on root and needle feeding insects using Beauveria positive
39
and endophyte free pine seedlings.
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Results. Beauveria bassiana was detected in seedlings which had not previously been exposed to
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the fungus, indicating a vertical mode of transmission. The fungus could colonise all parts of the
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pines, but did not always persist. We found that the presence of the fungus negatively affects the
43
fitness of the below-ground insect feeding on the plant by reducing their survival by over 10% and
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their weight by 5%. This study also showed that the mode of action of endophytic B. bassiana in
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pine is likely to be by feeding deterrence of insects induced locally by fungal metabolites, rather
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than by direct fungal infection of the insects.
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Discussion. A vertically transmitted beneficial endophyte of pine could be used as a cost effective
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approach to control insect pest in these commercially grown trees.
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KEYWORDS: endophytes, Beauveria spp., Pinus radiata, Brassica, biological control
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SUGGESTED RUNNING HEAD: Beauveria bassiana as an endophyte of Pinus radiata
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INTRODUCTION
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Pinus radiata D. Don (Pinales: Pinaceae) is a fast-growing softwood intensively used in the forest
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industry around the world (Mead 2013). This species is the most extensively planted production
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tree in the New Zealand (New Zealand Ministry of Primary Industry 2013). The economic value
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of this tree, denotes the need to consider both the biological and socio-economics aspects for its
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management (Mead 2013). This tree is highly susceptible to a range of ravaging insect species,
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comprising both above-ground and below-ground feeders.
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In the light of the importance of integrated pest management strategies implemented in New
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Zealand, biological control of P. radiata’s pests by natural enemies should be investigated. With
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this consideration, one possibility would be the use of the common terrestrial insect pathogenic
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fungi Beauveria spp. Vuillemin (Ascomycota: Hypocreales), which occurs on multiple hosts in
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most regions of the world (Bissett & Widden 1988, Glare & Inwood 1998, Glare et al. 2008,
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Rehner & Buckley 2005, Rehner et al. 2011, Roy et al. 2010). The life cycle and infection pathway
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of insects by these fungi is already well characterised (see Tanada & Kaya 1993). Recently one
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species belonging to this genus, B. bassiana (Bals.) Vuill., has also been reported to be capable of
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becoming endophytic in a variety of plant species (Akello et al. 2007; McGee 2002; Ownley et al.
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2008; Ownley et al. 2010; Powell et al. 2009; Quesada-Moraga et al. 2009; Reay et al. 2010; Tefera
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and Vidal 2009; Vega 2008), including in P. radiata (Reay et al. 2010, Brownbridge et al. 2012)
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and P. monticola (Ganley and Newcombe 2006). There is some evidence that while acting as plant
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endophyte, Beauveria can reduce pest damage (Gurulingappa et al. 2010; Qayyum et al. 2015) by
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inhibiting insect development and establishment (Bing & Lewis 1991, Cherry et al. 2004, Ownley
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et al. 2004, Vega et al. 2008). It can also be antagonistic to plant pathogens (Ownley et al. 2008,
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Ownley et al. 2010).
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In 2010, Reay et al., using culture-based recovery methods, reported the natural occurrence of B.
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bassiana in the seed of a single P. radiata pine cone and the recovery from needles and roots of
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about 15% of mature trees tested, suggesting that the fungus could be vertically transmitted. This
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mode of transmission has previously been shown for opium poppy Papaver somniferum (Quesada-
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Moraga et al. 2014). It has been established that endophytic infection by Beauveria may occur at
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various stages of the plant’s growth and may be introduced into the host plant from either the
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rhizosphere or the phyllosphere (Gomez-Vidal et al. 2006; Gurulingappa et al. 2010; Tefera and
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Vidal 2009). It has also been shown that B. bassiana could be established in P. radiata seedlings
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by either coating the seeds or root dipping, however there was very limited persistence of the
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fungus in the plant after 9 months (Brownbridge et al. (2012).
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So far, it is unknown whether the endophytic colonisation of pines by B. bassiana is beneficial to
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its host (Brownbridge et al. 2012). The present study aimed to evaluate the prevalence of
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endophytic B. bassiana in the Monterey Pine P. radiata, and to test whether the occurrence of the
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fungi negatively affects the fitness performances of above and belowground insect pests. The
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invasive corn earworm Helicoverpa armigera (Hübner) and larvae of the New Zealand beetle
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Costelytra zealandica (Scarabaeidae), two substantial generalist phytophagous pests, were
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respectively chosen as above- and below-ground insect feeders to carry out the experiments in this
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study.
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MATERIAL AND METHODS
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1. INVESTIGATION OF BEAUVERIA BASSIANA TRANSMISSION IN PINUS
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RADIATA
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1.1. Establishing background infection rates in seed
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This study used Pinus radiata seedlings grown from commercially obtained seed (PF Olsen seed,
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GF Plus; Amberley & Kaingora and Seddon, NZ). Experiments commenced in January 2012. To
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determine if Beauveria was present in the seed source, thirty seeds were surface sterilised (1 min
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70% EtOH, 7 min 0.04% NaClO, washed 2 x 1 min sterile distilled H2O), and grown on 1% water
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agar media in sterile conditions for approximately 10 days at 22°C. Once seedlings were
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approximately 6 cm in length, the plants were cut into 2 cm sections and placed on Beauveria
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semi-selective media (BSM) plates (consisting of: quarter strength PDA (Difco), 350 mg/L-1,
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streptomycin sulphate, 50 mg/L-1 tetracycline hydrochloride and 125 mg/L-1 cyclohexamide)
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(Brownbridge et al. 2012). These plates were incubated for 21 days at 20°C in the dark. A further
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twenty seeds were sown according to the same procedure in order to obtain pure pine DNA from
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newly emerged seedlings grown in sterile conditions. From the cultured plates, emerging fungal
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colonies were identified by microscopic examination. Any potential Beauveria spp. colonies were
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transferred to fresh medium (PDA, Difco) and maintained as pure cultures.
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1.2. Identification of seed-line recovered Beauveria
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Samples from pure fungal cultures on PDA were used to inoculate 10 ml of PDB (Difco), grown
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24-48 hours at room temperature (~22°C) and shaken at 160 rpm. Hyphae was filtered from
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cultures and ground to powder using liquid nitrogen and DNA was extracted using a Plant
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Genomic DNA Extraction System (Viogene, Taiwan) following the manufacturer’s instructions
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and re-suspended in 200 µl of water. The DNA was diluted 1:10 in water and then used for PCR.
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DNA was isolated from pure fungal cultures and amplified for a fragment of the translation
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elongation factor (TEF1-α) gene using the primers EF349 (5'- TGGCCACCAGCACTCACTAC)
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and EF1685R (5'- ATGTCACGGACGGCGAAA), designed to amplify an internal fragment of
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approximately 1350 bp (Reay et al. 2010). PCR amplification was performed in 25 µl volumes
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consisting of 0.7U/reaction Fast start polymerase (Roche), buffer, 2mM MgCl2, and 0.8 µM of
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each primer. Amplifications were carried out in a thermal cycler using 40 cycles of 45 sec at 95C,
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45 sec at 55C and 2 min at 72C. Positive and negative (sdH2O) controls were included in each
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PCR run. PCR products were visualised on a 1% TAE-agarose gel to check size and purity, and
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then sequenced with an ABI 3130xl (Applied Biosystems) DNA sequencer to confirm identity
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(Lincoln University Sequencing Unit, New Zealand). Sequences were checked for identification
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also using BLASTn (Altschul et al. 1990) and aligned with sequences obtained from previously
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published phylogenies (Rehner et al. 2011) to confirm species.
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1.3. PCR detection of Beauveria in planta
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Primers designed to amplify 431 bp of the translation elongation factor 1-α gene (TEF1-α) were
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optimised specifically for direct detection of endophytic Beauveria sp. from P. radiata DNA
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samples in one-step PCR experiment (primer sequences as follows EF52F 5’- 3’:
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TGACAAGCTCAAGGCCGAG and EF481R 5’ – 3’: GGAGGGCTCAAGCATGTTGT).
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Amplifications were carried out in a thermal cycler using 40 cycles of 45 sec at 95C, 45 sec at
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60C and 1:30 min at 72C. The reaction set-up was otherwise the same as described above. Pure
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P. radiata DNA was also included in each run. PCR products were visualised on a 1.2% TAE-
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agarose gel and then sequenced. Sequences were checked for identification also using BLASTn
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and aligned with TEF1-α sequences obtained from the Genbank (NCBI) database and local
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(unpublished) genome projects in order to conduct a phylogenetic analysis. Nucleotide alignments
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were performed in Genious Pro ver. 5.6.5. (Biomatters Ltd, NZ) using the Clustal W. algorithm
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with default parameters and phylogenetic analysis was conducted in MEGA 5.05 (Tamura et al.
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2007) . The evolutionary distances were computed using the Maximum Composite Likelihood
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method (Tamura et al. 2004) and were in the units of the number of base substitutions per site. The
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final analysis involved 15 nucleotide sequences. All positions containing gaps and missing data
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were excluded. There were a total of 260 positions in the final dataset.
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All pine DNA samples were simultaneously amplified with the universal plant primers psbA3_f
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(Sang et al. 1997) and trnHf_05 (Tate and Simpson, 2003) with the same reaction set-up but
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according to the following protocol: 35 cycles of 30 sec at 95C, 30 sec at 65C and 1 min at 72C.
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The sensitivity of the PCR detection assay was tested against a serial dilution of B. bassiana DNA
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spiked into pure plant DNA and sdH2O. The DNA concentration was ascertained using a Nanodrop
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spectrophotometer 3.0.0. and the number of genome/TEF1-α gene copies calculated per microliter
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of sample according the instructions supplied by Applied Biosystems for ‘creating standard curves
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with genomic DNA’ (http://www6.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf).
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Using this dilution series as gDNA template, the EF52F and EF481R primers were able to amplify
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as little as 30 copies of the Beauveria TEF1-α gene, with bands visualised on 1.2% agarose gel in
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TAE.
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1.4. Visualisation of endophytic fungi with transmission electron microscopy
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To visualise the fungus in seedlings, the direct PCR detection method was used on the DNA from
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the twenty seedlings germinated from seed to determine those samples to fix for transmission
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electron microscopy. Each germ tube was divided into two sections and one half surface sterilised
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and DNA extracted as described above. Using the primers EF52F and EF481R, each pine DNA
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sample was checked for the presence of Beauveria. As the seed had not been exposed to Beauveria
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prior to extraction, positive detection of Beauveria likely represented naturally resident
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endophytes. One germinated seedling was positive for Beauveria. The remaining section of the
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germ tube was fixed for TEM by immersion for 2 hours at room temperature (RT) in 2%
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glutaraldehyde/3% formaldehyde in 0.1M phosphate buffer (pH 7.2), followed by 3 washes in
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0.1M phosphate buffer (pH 7.2) and then stored in 1% formaldehyde in 0.1M phosphate buffer pH
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7.2 at 4oC. Samples were processed in 1% osmium tetroxide in 0.05M phosphate buffer (pH 7.2)
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at RT for 1, 2 and 4 hours, then washed three times in 0.05M phosphate buffer. Dehydration was
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through an acetone series at RT, then embedded in an epoxy resin (Procure 812, ProSciTech Pty,
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Australia) at 60oC for 22 hours. Ultrathin sections (90 nm) were cut using diamond knife
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(Diatome-US Pennsylvania USA) fitted in an ultramicrotome (Ultracut UTC, Leica Mikrosysteme
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GmbH, Vienna, Austria.) collected on Formvar coated 100 mesh copper grids. Sections were
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stained with uranyl acetate followed by lead citrate and examined with a Morgagni 268D TEM
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(FEI Company, Oregon, USA) operating at 80kV and fitted with a 40 µm objective aperture.
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Images were captured digitally.
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2. EFFECT OF ENDOPHYTIC BEAUVERIA BASSIANA ON INSECTS IN PINUS
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RADIATIA
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2.1. Model system
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Two insect species were used to determine the effect of endophytic Beauveria to root and needle
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feeding insects. Third instar larvae of the New Zealand grass grub, Costelytra zealandica, were
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collected from a dairy pasture recently converted from a pine forest in the North Island of New
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Zealand (Tokoroa, 38o15’36.74”S 176o00’35.21”E). First instar caterpillars of Helicoverpa
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armigera were obtained from Plant & Food Research, Auckland (Ann Barrington). Thirty-five
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pine seedlings were grown and monitored for the presence of Beauveria endophytes over the
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course of two years. Trees were cultivated in a glasshouse for the entire duration of the study and
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grown in 12-14 months old potting mix comprised of 80% composted bark, 20% pumice grade 1-
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7mm, 5g/L osmocote extract, 1g/L horticultural lime and 1g/L hydraflo wetting agent. The potting
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mixes were also screened for the presence of B. bassiana prior to use by plating on the semi
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selective medium.
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2.2. Detection of endophyte in two year old P. radiata trees
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To determine the presence of Beauveria sp. endophytes, small sections of young roots and needles
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were sampled from each pine tree, rinsed under cold water and stored individually at 4°C. From
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these samples, a 5 cm long fragment of root was surface sterilised (5 min in 2.5% Sodium
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hypochlorite, rinsed in sterile distilled water (sdH2O), then 1 min in 70% ethanol and washed x 3
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in sdH2O). Sterilised root/needle samples were rolled onto a PDA medium plate to assess the
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efficacy of the surface sterilisation procedure (Schulz et al. 2011). The PDA plates were incubated
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at 25°C for three weeks. No fungal cultures were observed from these plates. Root and needle
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sample were snap frozen and grounded in a sterile mortar. DNA extractions were then performed
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using a Viogene DNA/RNA extraction kit following the manufacturer protocol. DNA extracts
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were used in PCR to ascertain which trees were positive for endophytic Beauveria. The trees were
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subsequently used as either control pines (if no B. bassiana endophyte was detected, i.e. there was
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< 30 copies TEF1-α per 2 μL DNA) or treatment pines (B. bassiana was detected, or > 30 copies
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TEF1-α per 2 μL DNA) in the subsequent experiments.
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2.3. Effect of endophytes on below-ground insects
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Costelytra zealandica larvae (n=96) were weighed on a 0.01 g portable digital scale and their initial
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weight recorded. Subsequently, using a block design by initial larval weight (i.e. block 1: 0.14 g,
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block 2: 0.15 g, block 3: 0.16 g and block 4: 0.16 g), larvae were allocated to two different feeding
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treatments and kept in individual cells in multi-wells cell culture plates at 15°C. Half of the larvae
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were fed ad libitum with chopped roots from treatment pines (i.e. endophytically infected with B.
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bassiana), while the other half were fed with chopped roots from control pines (i.e. containing no
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endophytes). Fresh roots were added to the cells twice a week over five weeks. Larval survival
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and growth measured as weight gain were recorded as measures of fitness. Larval mortality rates
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were recorded weekly. The final weight of each larva was recorded at the end of the experiment.
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Statistical analyses on the effect of the endophytic occurrence of B. bassiana in P. radiata on larval
236
survival were carried out by Chi square testing. Larval growth was analysed by analysis of variance
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(ANOVA, complete block design) after exclusion of the larvae that died before the end of the
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experiment. Statistical tests were conducted with R version 2.12.1 (R Development Core Team
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2011) and GenStat® version 14.1 (VSN International, Hemel Hempstead).
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2.4. Effect of endophytes on above-ground insects
Helicoverpa armigera caterpillars (n=80) were weighed on a 0.0001 g readability digital scale and
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their initial weight recorded. A block design by initial weight (i.e. block 1: <0.0050 g, block 2:
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0.0050 g to 0.0079 g, block 3: 0.0080 g to 0.0109 g and block 4: ≥0.0110 g) was then followed to
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allocate the caterpillars to two different feeding treatments. Each caterpillar was kept individually
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in a 35 mL plastic container containing a small pine cuttings of 30 mm long placed in 2 mL
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eppendorf tubes filled with tap water to ensure that the cuttings remain turgid for the duration of
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the experiment. Containers were kept in an incubator on a 16:8 L:D cycle, at 23°C and 55%
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humidity. Half of the caterpillars were given cuttings from treatment pines (i.e. endophytically
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infected with B. bassiana), while the other half were fed with cuttings from control pines. Pine
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cuttings were replaced twice over the two weeks experiment to ensure an ad libitum access to food
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for the caterpillars. Caterpillar mortality and growth in term of weight gain were recorded at the
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end of the two weeks experiment. Statistical analyses on the effect of the endophytic occurrence
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of B. bassiana in P. radiata on caterpillar survival were carried out by Chi square testing. The
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growth of the caterpillars was analysed by analysis of variance (ANOVA, complete block design).
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Statistical tests were conducted with R version 2.12.1 (R Development Core Team 2011) and
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GenStat® version 14.1 (VSN International, Hemel Hempstead), respectively.
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RESULTS
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Electron microscopy
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A single emerged seedling out of twenty was positive for Beauveria and processed for TEM.
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Ultrathin sectioning showed fungal hyphae (Figure 1). Two to three hyphal strands were found
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intercellular in the pine samples (Figure 1). No fungus grew from the germinated seedling
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section placed on semi-selective BSM.
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Figure 1. Sections through a Pinus radiata germinating seed showing putative Beauveria sp.
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hyphae. A. Thin section stained with toluene blue, with hyphae in intercellular spaces (red
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arrows) (bar = 200 µm). B. Close up on thin section (bar = 50 µm). C, D and E. TEMs of
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hyphae (bar = 10µm, 2 µm and 0.2 µm).
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Endophyte detection in P. radiata
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Two Beauveria bassiana (sensu stricto clade A, Rehner and Buckley, 2005) colonies were isolated
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from the green tissues of newly emerged seedlings (designated isolates’ BPRC-F1 and BPRC-F2).
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For the 35 two-year-old pine trees tested, the presence of B. bassiana was detected by PCR
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(confirmed by genotyping) in two trees (trees PRO 11 and PRO 18, Figure 2). These two trees
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were subsequently used as the treatment pines in the two experiments of this study. The soil in
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which the trees were grown contained no detectable Beauveria species. For the Neighbour-Joining
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analysis, the optimal tree with the sum of branch length (= 0.2288) is shown in Figure 2. The
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percentage of replicate trees in which the associated taxa clustered together in the bootstrap test
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(1000 replicates) are also shown next to the branches (Felsenstein 1985). The tree is drawn to
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scale, with branch lengths in the same units as those of the evolutionary distances used in order to
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infer the phylogeny.
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Figure 2. Phylogeny of TEF1-α partial sequences of various fungal species (constructed with the
287
Neighbor-Joining method). The endophyte positive P. radiata samples are shown here aligning
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with B. bassiana (PRO 11 and PRO 18). A Beauveria isolate obtained from the same seed-line
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(BPRC-F2, identical to BPRC-F1) is also shown. Isolates used for comparison are either GenBank
290
obtained (gi|..) or the Lincoln University fungal collection. 2860 =ARSEF 2860 from Genbank
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ascension ASM28067v1.
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Effect of endophytes on below-ground insects
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Belowground insect larvae fed with roots free of endophyte survived significantly more than those
295
fed with roots endophytically infected with B. bassiana (χ2 = 3.3758, d.f. = 1, p = 0.06616) (Figure
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3a). Although the average weight of the larvae tends to decrease over time under both treatments,
297
it was quite noticeable that the weight of the larvae decreased even more (by almost 5%) when fed
298
with roots endophytically infected with B. bassiana (ANOVA, F1,86 = 10.86, p < 0.001) (Figure
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3b).
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Figure 3. a) Costelytra zealandica larval survival with green indicating the larvae fed with pine
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roots free of endophyte and red indicating the larvae fed with pine roots endophytically infected
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with Beauveria bassiana. b) Larval weight variation with green indicating the larvae fed with pine
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roots free of endophyte and red indicating the larvae fed with pine roots endophytically infected
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with Beauveria bassiana.
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Effect of endophytes on above-ground insects
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Caterpillars fed on pine needles from treatment pines (i.e. endophytically infected with B.
309
bassiana) did not show any impairment in their growth (ANOVA, F1,66 = 1.03, p = 0.315) (Figure
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4) nor survival (χ2 = 0.5008, d.f. = 1, p = 0.4792) (Figures 5) compared to the caterpillars fed on
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pine needles from control pines.
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Figure 4. Individual Helicoverpa armigera caterpillar weight gain (black dots) and average weight
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gain per treatment and per block (red and green squares), with green indicating the caterpillars fed
315
with pine needles free of endophyte (C) and red indicating the caterpillars fed with pine needles
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endophytically infected with Beauveria bassiana (T1).
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Figure 5. Helicoverpa armigera caterpillar survival with green indicating the caterpillars fed with
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pine needles free of endophyte and red indicating the caterpillars fed with needles endophytically
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infected with Beauveria bassiana
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DISCUSSION
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Evidence that B. bassiana could be vertically transmitted has only been demonstrated in poppy
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recently (Quesada-Moraga et al. 2014). Our study supports that vertical transmission of Beauveria
326
as an endophyte may also occur in Pinus radiata. While there was no PCR detection of Beauveria
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from ungerminated seed directly, Beauveria was detected in newly emerged seedlings that had no
328
prior exposure to the fungus. Direct observation with TEM of a positive seedling showed fungus
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present in very small quantities, only 2-3 hyphal strands per transverse section. We failed to detect
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Beauveria in the majority of the young seedlings from surface sterilised seed and from the seed
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itself, using the PCR method. However, based on the observations made from the TEM images it
332
is likely that Beauveria can be present in very small quantities in seed. The seed casing may also
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contain some inhibitors to the PCR reaction (although seed DNA did amplify with pine specific
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primers, Glare, personal observation).
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Our study also suggests that plant host endophytic infection by Beauveria is relatively rare in
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nature, as B. bassiana was isolated from only two pine seedlings, perhaps indicating vertical
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transmission. However, a similar genotype of Beauveria was detected two years later from mature
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plants sown from the same seed batch. This is consistent with another study, which used root
340
inoculation, where Beauveria endophytes were established in pine seedlings from both inoculated
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seeds and roots independently, but in this instance, only one plant of thirty was positive for B.
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bassiana as an endophyte by 9 months (Brownbridge et al. 2012).
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So far, only a few studies on B. bassiana as plant endophyte have demonstrated effects of this
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fungal association against phytophagous insect (e.g. Bing & Lewis, 1991, Cherry et al. 2004,
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Powell et al. 2007, Akello et al. 2008a,b, Quesada-Moraga et al. 2009, Gurulingappa et al. 2010,
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Biswas et al. 2013, Akutse et al. 2013). Using C. zealandica and H. armigera as below- and above-
348
ground insect feeders, this study has shown that, when present as an endophyte in P. radiata, the
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fungus B. bassiana significantly reduces the fitness of below-ground insect feeding on the plant.
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351
In endophytic fungi-plant systems, some studies have shown the reduction in both feeding and
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growth of the insect after feeding on infected plants. For instance, a study by Griesbach (2000),
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with Fusarium sp. as plant endophyte shown that the larvae of the Banana Weevil Cosmopolites
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sordidu (Germar) (Coleoptera: Curculionidae) obtained from endophyte-inoculated plants were
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smaller in size than those obtained from non-inoculated plants. Similarly Gurulingappa et al.
356
(2010) reported a reduction in feeding in the cotton aphid Aphis gossypii Glover on cotton
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endophytically infected by B. bassiana. This aligns with the significant loss of weight observed in
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the larvae fed with pine infected with B. bassiana in the present study.
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Other studies suggest that the mode of action of B. bassiana as plant endophyte against insects
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results from direct mycosis infection of the insect by the fungus. For instance, Akello et al. (2008),
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who studied the effect of endophytic B. bassiana in banana plants on the banana weevil, C.
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sordidus, reported dead mycosed insects in the rhizome of B. bassiana-inoculated plants,
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suggesting a direct mode of action through mycosis infection of the banana weevils by the fungus.
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Nonetheless, in our study, none of the dead insects recovered from the feeding treatments with
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infected pines displayed B. bassiana mycosis.
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In a majority of studies, no fungal growth was observed on the dead body of the insects, leading
369
authors to hypothesize that the mode of action of B. bassiana might rather be due to the feeding
370
deterrence action induced by fungal metabolites or mycotoxins (Vega, 2008, Mantzoukas et al.
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2015) than by direct mycosis infection. This hypothesis is concordant with our results and would
372
also explain the significant decrease in weight observed in the larvae fed with B. bassiana-infected
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pines.
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It is important to note that the overall loss of weight itself, as observed in the larvae across both
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feeding treatments, could be due to (1) the advanced age of the larvae at the time of the experiment,
377
(2) the low nutritional value of the pines for the larvae and/or (3) the suspected existence of host-
378
races in C. zealandica (Lefort et al. 2014). Indeed, C. zealandica usually undergo a non-feeding
379
pre-pupation stage (East & Kain 1981, Wright 1989). This experiment was performed with larvae
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already weighting between 0.14 and 0.16g. These larvae might have reached their optimal weight
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to enter pre-pupation during the course of the experiment, hence cease feeding at an early stage of
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the trial, which would have resulted in a generalised loss of weight similar under both feeding
383
treatments. Furthermore, the nutritional values of the host plant on which C. zealandica larvae fed
384
can result in variations in fitness performance of the larvae (Lefort et al. 2015a, Lefort et al. 2014).
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It would seem that while C. zealandica is able to use P. radiata as a host, this plant might not offer
386
all the nutritional qualities required for optimal growth of the insect. Finally, Lefort et al. (2014)
387
suggested the possible occurrence of several host-races in this species, this provides a tangible
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explanation as to why the larvae used in this experiment, which were collected from a pasture, did
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not gain weight while feeding on pines.
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While in our study a feeding deterrence action appears as the best hypothesis to explain the loss of
392
weight of C. zealandica, our results also suggest that this effect against insect feeders remains
393
limited to the infected tissue as we did not detect any deterrent effect on the caterpillars fed with
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the needles of pines for which the roots were tested positive with the fungi. This implies that the
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mode of action of B. bassiana as plant endophyte might not be the result of cascading reactions
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occurring through the plant by systemic acquired resistance (SAR) but might rather be a localised
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response. The detailed chemical nature of fungi-insect-induced plant defenses remains poorly
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studied and sparse attention has been given to defense induction and SAR in plant root systems
399
(Erb et al. 2012, Pierre et al. 2012, Lefort et al. 2015b). Nonetheless, if the mode of action of plant
400
endophytic B. bassiana was through SAR, a negative effect on the fitness of the caterpillars fed
401
with needles would have been expected in addition to the one observed on the larvae fed on the
402
roots of the same plant.
403
404
In a study by Akello et al. (2008), endophytic B. bassiana was isolated from different parts of
405
maize plants, but to a greater extent from the roots and rhizomes of the plant. Furthermore, it
406
appears that B. bassiana cannot systematically be re-isolated from B. bassiana-inoculated maize
407
plants after several weeks post-inoculation (Akello et al. 2007, Akello et al. 2008). In the present
408
study, the molecular diagnostic for the occurrence of B. bassiana in the pine needle fed to
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caterpillars was performed several weeks after the diagnostic on the roots. Similarly, the
410
experiments using H. armigera caterpillars as above ground-feeder was performed several weeks
411
after the one on C. zealandica larvae. This raised further questions as to whether the mode of
412
action of B. bassiana occurs through SAR with a limited lifespan or is truly a localised response,
413
and on the persistence of the fungi in the plant.
414
415
The presence of B. bassiana as a vertically transmitted endophyte capable of reducing insect
416
damage could have implication for pest control in these long-life commercially grown trees. The
417
issues around longevity of colonisation would need to be overcome, but it is possible that a
418
vertically transmitted endophyte may provide a very long cost solution to forest protection from
419
insects.
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ACKNOWLEDGEMENTS
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The authors would like to thanks Jenny Brookes, Josefina Narciso, Narges Askari Nejad and
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Estelle Moreau (Bio-Protection Research Centre, New Zealand) for their technical support and
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Anne Barrington (The New Zealand Institute for Plant & Food Research Ltd, New Zealand) for
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providing the caterpillars used in this study. Richard Townsend (AgResearch Ltd, New Zealand)
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for supplying grass grub larvae. Duane Harland and James Vernon (AgResearch, Lincoln, NZ)
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provided expert assistance with the transmission electron microscopy. We also thank Prof Leo
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Condron (Lincoln University) and Dr Stephen Reay (AUT) for advice on the project.
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