Recipient Organization
POPLAR INNOVATIONS INC.
148 CORDMAR DR
CASTLE ROCK,WA 98611
Performing Department
(N/A)
Non Technical Summary
Hybrid poplar is the predominant woody perennial being developed for the bioeconomy as a purpose-grown source of cellulosic feedstock. P. deltoides × P. trichocarpa has been the most productive hybrid in the Northwest while P. deltoides × P. maximowiczii is likely the Southeast's best hybrid. But durable disease resistance is the principal obstacle in continued commercial use of these hybrids. Breeding for resistance to Melampsora leaf rust, the main pathogen in the Northwest, has been difficult due to the multiplicity of rust pathotypes. In the Southeast, attaining resistance to the principal pathogen, Septoria stem canker, has been challenging given the disease's broad range of virulence. Our goal is the development of endophyte-mediated resistance technology to overcome these pathogens either by direct antagonism or by prompting host-resistance mechanisms. The proposal therefore responds to program area 8.1's priority to develop "... better methods [and] control strategies for combating ... pathogens that attack important woody species..." The objectives are: 1) identification of bacterial endophytes that are antagonistic to Melampsora and Septoria in laboratory and greenhouse assays and 2) evaluation of endophyte technology in hybrid trials in the Northwest and the Southeast. George Newcombe will provide expertise in endophyte selection and application. Anticipated results are improved disease control methods that augment genetic resistance. Phase I will be devoted to endophyte discovery and establishment of field trials. Phase II will complete the fields trials. Phase III's objective is the joint commercialization of hybrids and endophytes in the two regions.
Animal Health Component
50%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Goals / Objectives
Phase I technical objectives address the most pressing consideration in the commercialization of endophyte-mediated resistance (EMR), namely the correlation between lab assays and field results. There are four technical objectives to be resolved in Phase I for effective execution of the Phase II project.Successful endophyte selection and propagation- Objectiveiis to complete thein agarolaboratory assays ofSeptoriaantagonists and thein plantagreenhouse trials ofMelampsoraantagonists. Endophytes gotten from tree and grass seed have already been cultured and are being maintained at the George Newcombe lab in Moscow, Idaho. Objectiveialso encompasses the development of multiplication procedures for scaling the most promising endophytes for Phase I and Phase II field trials. Critical questions are:Is a 'biocontrol complex' that includesBacillus subtilis,and other closely related species, a robust endophyte source for antagonists of poplar's two main fungal pathogens in laboratory and greenhouse trials?How rapidly can endophytes be scaled to quantities sufficient for field trials?The lone technical risk is lab safety. We consider the risk minimal, but standard operating procedures will nonetheless be closely followed.2. Successful test plot establishment- Objectiveiiwill establish replicated test plots in two locations utilizing an experimental design to support objectivesiiiandivand Phase II's analytical priority. One test plot is established at Westport with nineP. × generosavarieties. A land lease with Mississippi State University to install a second test plot, during Phase I, at the Delta Research and Education Center (DREC) in Stoneville with nineP. deltoides×P. maximowicziiclonal varieties. All varieties are Poplar Innovations proprietary property. Technical risks of establishment will be mitigated by:Poplar Innovations' experience in poplar cultivation and plot management that hasproven successful over the past three decades. The Westport plot will be managed by Poplar Innovations' staff. The Stoneville plot will be cared for by DREC personnel who will be guided by Poplar Innovations.Risk factors are wind throw, defoliating insects,and unseasonable drought. Annual coppicing will minimize wind throw at Westport. Irrigation is available at both locations to minimize the drought risk. Cottonwood leaf beetle isexpected at Stoneville, but frequent monitoring and timely applications of insecticide will minimize the defoliation risk.3. Successful endophyte inoculation protocol- A Phase I priority is to determine whether the endophyte inoculation procedure is dependable for Phase II work in which the Westport trial will test for the difference in Melampsora leaf rust infection between treated and control plots while the Stoneville trial will test for differences in Septoria infection. Two technical questions will be considered for objectiveiii:Following an aqueous spray of the bacterium preparation, can the endophyte inoculum be confirmed in treated plots but not in the control plots (i.e. no unintended drift)?Will the volume and distribution of the endophyte spray applied to the field trials during Phase I be adequate or will adjustments be needed?Successful disease evaluation protocol- The objective is to ensure Poplar Innovations has a thorough procedure for Phase II evaluation. Both the Westport and Stoneville trials will be assessed for the EMR effect using a combination of standard and novel evaluation techniques. The technical question is:Can proficiency be developed in using novel evaluation techniques?
Project Methods
The Phase I work plan flows from the four technical objectives. Laboratory trials will be conducted by George Newcombe at the University of Idaho facilities in Moscow, Idaho. Field inoculation trials and practice with novel evaluation techniques will be conducted at Westport, Oregon and Stoneville, Mississippi by Poplar Innovations.Technical objective i - Successful endophyte selection and propagation. Approach: Our project incorporates two very important features in trials to identify the most promising endophytes: 1) a focus on Bacillus subtilis as the featured endophyte and 2) its extraction and isolation from seeds. Empirically, we know that seed microbes are highly antagonistic to plant pathogens. Moreover, we know that B. subtilis, can not only be isolated from plant seeds, but it is extremely combative. These two factors - Bacillus endophytes sourced from seeds, will be a major step forward in the biocontrol field. Septoria endophytes will be identified in in agaro assays given Septoria's amenability to agar culture while Melampsora assays will be conducted in planta in a greenhouse.Tasks: Conduct challenge assays, select strains, multiply bacterial cultures for field trials.Methodology: Septoria assays will place the inoculum of the putative antagonist(i.e., one of many isolates of seedBacillus) twocm from inoculum of Septoria on a fresh agar plate. The effect of the putativeantagonists on Septoria growth will be calculatedas the percentage of growth relative to controls. Mean values will be calculatedfor three replicates. Melampsora in planta assays will involve replicated trials of susceptible poplar genotypessprayed with isolates of seedborneBacillusand inoculated s with rust urediniospores.Assessment will be made 14 days post-inoculation and expressed as uredinia per unit of leaf area. Trees will be inoculated with each putative antagonist without rust or with rust without an antagonist, or with neither rust nor antagonist. Scale up propagation will involve growing the selected endophyte strains on soy medium.Technical objective ii - Successful test plot establishment. Approach: Two field trials using a factorial study design will be used to evaluate the joint effects of endophyte at two levels (treatment versus control) and clonal variety at nine levels. An epiphytotic will be created to augment natural pathogen pressure by introducing infected leaves and branches into the test plots. The Westport test will isolate the effectiveness of rust-antagonistic endophytes in mediating P. × generosa susceptibility to Melampsora in the absence of Septoria. (Septoria is not present at Westport.) Likewise, the Stoneville test will isolate canker-antagonistic endophytes in mediating P. deltoides × P. maximowiczii susceptibility to Septoria. Though rust is present at StonevilleP. deltoides × P. maximowiczii varieties show durable rust resistance in Mississippi.The existing Westport trial will be coppiced at the end of the 2023 growing season for Phase I and Phase II studies. The Stoneville trial will be planted at the Delta Research and Education Center (DREC) in 2024 and will not be coppiced through Phase II to allow the development of perennial stem cankers. The experimental design will be a randomized complete block with four replicates. The treatment design will be a split plot factorial with the endophyte factor assigned to the whole plots. The clonal variety factor will be represented by single-tree subplots to increase the precision of detecting varietal-by-endophyte interactions. Whole plots will be double buffered on all sides with isolation alleyways to preclude drift of the inoculum spray to the control plots. Spacing will be 2 m × 4 m.Tasks: The Westport trial has 100% survival; trees that fail to coppice in the trial will be replanted. Sufficient hardwood cuttings will be collected for the Stoneville trial from the Westport clone bank to double plant the trial to ensure 100% survival. Cuttings will be treated before shipment if specified by a Mississippi State phyto-sanitary permit.Methodology: The only differences between the two trials will be the composition of the endophyte treatment and test varieties: 1) rust-antagonistic endophytes and P. × generosa varieties at Westport and 2) canker-antagonistic endophytes and P. deltoides × P. maximowiczii varieties at Stoneville. The varieties being used in the Northwest trial are ones that have shown moderately high or very high levels of rust infection in previous P. × generosa plantings at Westport. P. deltoides × P. maximowiczii varieties are ones that have mostly shown cankering in previous trials in Mississippi.Technical objective iii - Successful endophyte inoculation protocol. Approach: We will test that our endophyte-inoculation protocol can be applied without drift to the control plots. We will use an aqueous spray solution containing endophytes for the treated plots and the same solution lacking endophytes in the control plots. Westport and Stoneville trials will be treated in July and September.Tasks: A pressurized hand sprayer will be used. The volume of the spray mixture will be based on a calculation of canopy area and interviews with area orchardists. The endophyte concentration will be based on a G. Newcombe recommendation. Application to the trees will be made by spraying atop from an orchard ladder and below from the ground. (The same spray mixture without the endophytes will be applied to the control plots.) Whole plots will be sampled to determine whether the endophyte spray has drifted to the control plots.Methodology: Abaxial and adaxial leaf surfaces and stems will be treated. Agar plates will be placed in the treatment and control plots just prior to spraying. The number and viability of colony-forming units will be assessed by reading the plates 72 hours later to determine whether spray drift from the treated to the control plots has occurred. Technical objective iv - Successful disease evaluation protocol. Approach: Main effects and interactions will be assessed in Phase II using a range of response variables specific to the two regional trials and pathogens. Phase I will be used to gain expertise with those response variables for which Poplar Innovations has not extensive experience.Tasks. Two measurement types will be taken. First will be morphological phenotypes that may be associated with the effectiveness of endophyte colonization and/or the rate of pathogen infection. These data will be collected prior to the appearance of disease symptoms to gain insight into why different varieties may or may not be responding to the endophytes. Second will be those phenotypes that are expressed after disease has run its course to ascertain the effectiveness of EMR control.Methodology: Leaves from the 5th plastochron index will be collected prior to the onset of disease symptoms from each variety at the test sites and measured for stomatal density and specific leaf area. Also bark thickness and sylleptic branching will be measured at Stoneville. Morphological data will be analyzed to see if they condition varietal-specific responses to the endophytes.During the height of the rust and canker infection periods, the following response variables that will be evaluated:Healthy, chlorotic, and necrotic tissueCrown areaTiming of leaf fallScores of rust and leaf spot scoresCarbohydrate content of stem tissueCold hardiness by electrolyte leakage testCounts of stem and branch lesions and length of lesions.