Item No. 1 of 1

INVESTIGATOR: DeWalt, S.

PERFORMING INSTITUTION:
CLEMSON UNIVERSITY
CLEMSON, SOUTH CAROLINA 29634

GENOTYPIC SPECIFICITY IN PLANT-HERBIVORE INTERACTIONS AND ITS APPLICATION TO MANAGEMENT OF AN INVASIVE WEED

NON-TECHNICAL SUMMARY: Optimal matching of host and pathogen genotypes may improve the efficacy of pathogens released as biological control agents against invasive weeds of agricultural weeds and managed woodlands. Depending on the degree of local adaptation of plants to their pathogens, foreign exploration for weed biological control agents may need to be focused on native provenances of particular genotypes of the invasive plant (i.e. the location from where the weed originated), specifically directed away from the native provenance, or conducted wherever feasible. We are investigating the potential for optimizing future biological control of a globally significant weed using genetic surveys and host specificity trials. Clidemia hirta is recognized as one of the world's worst weeds and is among the gravest invasive threats to tropical island ecosystems and biodiversity. This invasive plant has been a target for biocontrol for decades, but agents released to date have not provided sufficient control. The geographic origins of invasive populations have never been pinpointed, so the potential for developing more effective biocontrol agents optimally adapted to the invasive genotypes is unknown. Close matching of invasive plant genotypes with coevolved natural enemies from their native range is generally thought to be important for successful biocontrol, but the effectiveness of deliberately exact genetic-matching is debated. This is important from a practical point of view because genetic surveys of invasive plants across their native ranges require time and effort, which might be used otherwise in discovery and testing of new biocontrol agents. By better understanding the benefits to be gained from genetic-matching of invasive plants and natural enemies, we will improve our efficiency and success in developing biocontrol.We are testing the utility of this approach with a particularly promising candidate agent, the newly described nematode Ditylenchus gallaeformans. This gall-forming natural enemy has been observed to severely impact growth and reproduction of various species within the plant family Melastomataceae in its native range. We have already conducted extensive genetic surveys across the native range of C. hirta and are doing so with the nematode. We will then compare nematode virulence and plant resistance for a variety of plant-nematode genotype combinations. Besides providing a test of the value of careful genotype-matching to biocontrol research in general, this project is expected to identify optimal sources of a highly effective agent for long-term management of this weed, which is of major importance in forests and agricultural lands of Hawaii, American Samoa, Palau, and other islands and ecosystems across the Pacific region. In addition, this project will serve as a test-case of control between a nematode and melastome plant and will be used to determine how control of other invasive plants in Hawaii and elsewhere may be achieved.

OBJECTIVES: The goal of this project is to address how important coevolution is between natural enemies and their plant hosts in determining resistance and tolerance of plants. Depending on the degree of local adaptation of plants to their herbivores and pathogens (i.e. natural enemies), foreign exploration for weed biological control agents may need to be focused on native provenances of particular genotypes of the invasive plant, specifically directed away from the native provenance, or conducted wherever feasible. We will address this question of coevolution by examining a tropical woody plant and a nematode species, which is a common natural enemy that is being pursued as a biocontrol agent. In addition to supporting development of this promising biocontrol agent against a highly invasive plant, this project will evaluate the general question of whether determining the genetic structure of invasive species and their proposed biocontrol agents in the native range is a necessary first step for developing a biological control program.The supporting objectives of the project are the following:1) quantify distribution of genetic variation in the host plant species and nematode across their native ranges,2) assess whether there are differences in pathogenicity and virulence of the different nematode genotypes on different host genotypes, and3) measure the benefit in terms of greater pathogenicity gained from optimal matching of plant and nematode genotypes.

APPROACH: In each of 8 sampling locations in the native range, we will travel extensively to find at least 20 individuals from which to collect leaves for the phylogeographic study and seeds for the pathogenicity study. In addition, we will collect from 20 individuals in Hawaii and Singapore, parts of the invasive range.For the plant phylogeographic study, a piece of leaf material from each maternal plant will be genotyped using nine microsatellite loci developed specifically for C. hirta (DeWalt, unpublished data). The geographic patterns in genetic similarity among these native populations will be examined using ordination of genetic distances among populations (the classic method) and an individual-based clustering method. For the nematode phylogeographic study, nematodes will be isolated from 10 individuals of C. hirta per collecting location. Nematode DNA will be sequenced at a portion of the mitochondrial cytochrome oxidase c subunit 1 (COI) gene to determine divergence among geographic regions. We will calculate standard measures of genetic variation, such as nucleotide diversity and gene diversity, and will explore intraspecific relationships between the observed haplotypes using a minimum spanning network. The hypothesis that there is genetic divergence in different regions will be tested by comparing the genetic distances among individuals collected in different geographic regions using multivariate methods. We will conduct two greenhouse experiments for pathogenicity trials. In the first experiment, we will use nematode inoculum sources cultured on their "native" genotypes. In the second, we will use sources cultured on the Hawaii or Singapore genotypes.For Experiment 1, we will collect naturally infected leaves and inflorescences from the number of plants described above and culture them in the Clemson University greenhouses on 10 plants grown from each of these 8 native locations. After at least eight months of culturing, nematode inoculum will be isolated from each of the rearing plants, combined from the 10 rearing plants from each collecting site, examined under a stereomicroscope, and standardized to 1500 nematodes of mixed stages per 1 mL. Each plant will be inoculated in one location - in the middle of the highest growing tip (C. hirta has an opposite leaf arrangement). We will evaluate plant resistance to nematodes after 3 mo, which is 2 mo after symptoms generally begin to appear Resistance will be evaluated using three measures. The first two quantify host plant susceptibility to nematode attack: first, we will measure symptom severity and, second, nematode reproduction. These two measures of resistance are often not well correlated. Third, we will use plant growth rates as a measure of each plant's ability to tolerate nematode attack. Symptom severity will be evaluated by counting numbers of leaves or growing points galled and removing each gall and measuring its wet weight. Percent of foliage affected by galling will be estimated visually for each plant. We also will photograph each plant from a standard distance and store photos to allow later scoring for degree of galling. Nematode reproduction will be determined. The effect of nematodes on plant growth will be evaluated by taking the difference between growth rates of inoculated plants versus nematode-free controls in terms of total stem length, basal diameter, aboveground biomass, and belowground biomass. A second trial of the experiment will be conducted after harvesting the first trial to be assured of consistency in the results. We will test whether symptom severity, nematode density, and tolerance differed among plant genotypes or inoculum source using an analysis of variance (ANOVA) looking at the fixed effects of experimental genotype and inoculum source. Using linear contrasts, we will determine whether this proposed biocontrol agent is more virulent on plants collected from the same or "home" vs. different or "away" collecting sites (e.g. the reproduction factor is greater for nematodes from Puerto Rico grown on plants from Puerto Rico than those grown on plants from all other native locations).For Experiment 2, we will use a similar design as Experiment 1, but we will rear all inoculum sources on the Hawaiian or Singapore genotypes rather than their "home" plant genotype and then apply them only to Hawaiian and Singapore genotypes. By growing all inocula on the two invasive genotypes, we can determine whether the nematodes can be cultured successfully on these invasive genotypes without losing their original pathogenicity and virulence. Ultimately, a successful biocontrol effort will need to grow the inoculum in quarantine facilities on living plants, as these nematodes do not survive on artificial media and nematodes maintained on fungal cultures may have reduced pathogenicity. The methods will be as described in Experiment 1. We will test whether symptom severity, nematode density, and tolerance differs among genotypes using ANOVA looking at the main, fixed effects of original inoculum source, rearing genotype (HI or SG), and experimental genotype (HI or SG). We will also look at interactions among these effects to determine: 1) whether inoculum from different collecting sites differs in its virulence between the two invasive genotypes, 2) whether the identity of the most virulent and least virulent nematode sources were the same in Experiments 1 and 2 (i.e., there is a significant effect of rearing host on virulence if the rankings differ), and 3) whether culturing the nematode on C. hirta is likely to work in quarantine facilities using the invasive genotypes.Efforts:We will publish our results in scientific journals that target biological control practitioners and weed scientists to convey which genotypes of the proposed biological control agent will be easiest to culture and most efficacious at controlling the weed. In addition, we will convey our results directly to biocontrol practitioners in Hawaii through presentations and reports.Evaluation:The success of the project will be measured by the ability to pinpoint from where in the native range we should go to collect nematodes for development as a biological control agent and a standard protocol for culturing the nematodes. The quality of the publications will also be a measure of success.

PROGRESS: 2015/01 TO 2020/01
Target Audience:We sponsored research by one female student from Brazil who is completing her doctorate at Clemson University. During the project, this student made contact and shared knowledge with weed scientists in several areas where Clidemia hirta is invasive, and worked together with plant ecologists and nematologists in Brazil and Trinidad, two countries within the plant/nematode native range. Also, during the research period, the graduate student attended several workshops that helped with her technical training, like bioinformatics, microscopy and microbiome analysis, and meetings, where she shared and discussed the results with other researchers. Changes/Problems:Due to the fact that D. gallaeformans, when artificially inoculated, did not establish or cause major infection or symptoms on C. hirta, we had to reviseobjectives #2 and #3. After exhaustively trying to grow this nematode in the greenhouse, we conclude that this species would be an unsuitable biological control agent. A suitable biological control agent would have to be able to be raised under a range of conditions similar to what is available in the Clemson University greenhouses. If we were to conduct host-specificity testing, we would never know whether the nematode failed to create galls because of the conditions or the host species. Nevertheless, we have determined that host-genotype specificity is unlikely in this plant pathogen given that nematode genotypes are not species specific. In addition, we learned much more about the invasive plant C. hirta, including that there are only two invasive genotypes and they are morphologically very distinct. The microbiome study we are now pursuing will further allow us to understand how galls may be made. What opportunities for training and professional development has the project provided?We supported a PhD graduate student from Brazil, who started on the project in August 2016 and will be graduating in August 2020. This student participated in bioinformatic trainings as well as workshops like laser capture microdissection and plant microbiome. We also supported a lab technician who is currently pursuing an MS in Plant and Environmental Studies. How have the results been disseminated to communities of interest?DeWalt has been in contact with biological control practitioners and weed scientists from around the world to bring attention to this species. DeWalt attended the PD meeting in Washington, D.C. at the end of October 2017 and presented a poster on the findings of the project to date. In addition, the graduate student presented our findings in regional meetings, poster presentations in symposiums, and international nematology meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

IMPACT: 2015/01 TO 2020/01
What was accomplished under these goals? To fulfill objective 1 to quantify distribution of genetic and morphological variation in the host Clidemia hirta, we conducted three studies: a widespread phylogeographic analysis using microsatellites, a morphological comparison of leaves from herbarium specimens, and a common garden experiment. For the phylogeographic/genetic work, we obtained leaf samples from 10 locations in the native range (Neotropics: Dominican Republic, Puerto Rico, Dominica, Costa Rica, Trinidad, Tobago, Guyana, Suriname, French Guiana, Brazil) and 13 in the invasive range (Asia/Australia: Singapore, Sabah, Sumatra, Java, Australia; Africa: Tanzania; Indian Ocean islands: Mauritius, La Reunion, Seychelles, Madagascar; Pacific Ocean islands: 4 islands of Hawaii, American Samoa, Palau, Pohnpei). We also isolated DNA from herbarium specimens and DNA directly provided by Fabian Michelangeli (NY Botanical Garden) to increase the breadth of sampling to include specimens from Jamaica, Cuba, Colombia, Guyana, Venezuela, Peru, and other locations in Brazil. We genotyped the samples at nine microsatellite loci and found that Clidemia hirta was genetically variable across the native range, but invasive range populations only had one of two multilocus genotypes. Of those two "invasive genotypes", one genotype was found in all populations in the invasive range with the exception of Sumatra and Java. We call this the "Hawaii genotype". We call the more restricted invasive genotype the "Singapore genotype" and only found it in Singapore, Sabah, Sumatra, and Java in the introduced range. None of the native range plants matched the "Hawaii genotype" exactly. However, the closest match was found in southeastern Brazil (Paraná state). We found a genetic match for the less common invasive "Singapore" genotype in two locations of the native range - Venezuela and the island of Dominica. We examined morphological variation in C. hirta from herbarium specimens collected from across the native range and conducted leaf shape and trichome counts on leaves from these accessions. Trichome densities and leaf shapes of plants from Central and South America and the Caribbean Islands were statistically indistinguishable. We then conducted a common garden experiment in a greenhouse at Clemson University over 13 months (November 2016 to December 2017) using seeds of Clidemia hirta seed collected from five parts of the invasive range (Singapore, Hawaii, Palau, Pohnpei, La Reunion) and six parts of the native range (Puerto Rico, Dominica, Costa Rica, Brazil, Trinidad, French Guiana). We measured the height and total stem length of these plants at various time points to examine growth rates. We also harvested leaves at different points to count the number of trichomes, conduct leaf shape analyses, and measure the specific leaf area (a measure of thinness or thickness of the leaves). No statistical differences were found in plant height, mass, number of trichomes, specific leaf area or other morphological variables between native and invasive range genotypes. However, morphologically the plants grown from seed from Brazil were indistinguishable to the eye from plants grown from seed from introduced range locations of the "Hawaii genotype" and were distinguishable from the "Singapore genotype" plants - those from Singapore. We are packaging the molecular work, the herbarium work, and common garden results into one manuscript to be submitted for peer review in Fall 2020. Our major accomplishments for the second part of objective 1 (to quantify distribution of genetic variation of D. gallaeformans) have been the following. Phylogenetic and genetic diversity studies were performed using COI mtDNA sequence fragments from nematodes from Costa Rica, Dominica, and Trinidad respectively. The sequences were aligned, and sequences from 14 nematode species, including other Ditylenchus spp., were added as outgroups. A maximum likelihood (ML) analysis was performed and, from this dataset, a pairwise distance matrix was generated to calculate intra- and interpopulation genetic divergence. The phylogenetic reconstruction showed that D. gallaeformans samples clustered together, forming a monophyletic group that were divided in 2 main clades. The first well-supported clade comprised the individuals from Costa Rica and Dominica. The second well-supported clade is a sister clade of the first clade and included sequences from Trinidad. This clade is composed of three subclades. However, the D. gallaeformans samples did not cluster together with the other Ditylenchus species added in the analysis, which means that the Ditylenchus genus is paraphyletic. The genetic distances observed were large enough to conclude that there are differences among populations but small enough that we are confident they belong to the same species. Haplotype networks among geographical locations and host species were congruent with the phylogenetic reconstructions, showing segregation of the mitochondrial haplotypes according to location but not host species. There was no haplotype sharing among countries. Thus, we conclude that there is genetic variation within D. gallaeformans due to geographical location but there does not appear to be specialization due to host species. A manuscript with these results will be submitted to Nematology in Summer 2020. The second and third objectives have been the most challenging because we were unable to successfully reproduce symptoms we see in the field. We inoculated different genotypes of C. hirta plants with different nematode genotypes in greenhouse and field trials. In the greenhouse at Clemson University, we mimicked conditions where we find the most galls in the native range, however infection failed to occur and galls as seen in the field failed to develop. After several unsuccessful trials to raise D. gallaeformans in the greenhouse, we conducted a field experiment in Trinidad, where this nematode is found causing galls on C. hirta. Nematodes were collected from naturally infected hosts and inoculated on healthy Melastomataceae plants in the field. We also transplanted plants from the field to pots, inoculated them and kept them in a greenhouse at the University of West Indies in Trinidad. Unfortunately, none of the approaches worked, meaning that the greenhouse conditions were not the major problem for nematode development. Considering that nematodes could not be successfully raised in greenhouse or field conditions, we have determined that D. gallaeformans would be an unsuitable biological control agent for C. hirta. A manuscript with these results will be submitted to Journal of Nematology in Spring 2020. The fact that we couldn't raise nematodes led us to wonder the basic question: how are galls formed? We have begun new experiments comparing the bacterial microbiome of galled tissue to non-galled tissue. The bacteria were our target because of their capacity to induce galls on plants. Preliminary results showed us that there are significant differences between the bacterial composition found in healthy leaves versus the ones in galled leaf tissue and there are also differences among host plant species.

PUBLICATIONS (not previously reported): 2015/01 TO 2020/01
No publications reported this period.

PROGRESS: 2017/01/15 TO 2018/01/14
Target Audience:We sponsored research by one female graduate student, made contact with weed scientists in several areas where Clidemia hirta is invasive, and worked with plant ecologists and pathologists throughout the native range of C. hirta. Changes/Problems:We have had a major problem determining the ideal rearing conditions for the nematode. As mentioned above, we will consider moving our experiments to Trinidad if we are able to get galls to develop there. If the field application of inoculum in Trinidad does not work in this latest experiment, we will have to substantially revise objectives #2 and #3. What opportunities for training and professional development has the project provided?We continued to employ a full-time technician until January 2018, and that person had the responsibility for taking care of the plants in the common garden and after inoculation. She started a MS degree in Spring 2018 in the Plant and Environmental Studies program. We also supported a PhD graduate student from Brazil, who started on the project in August 2016. How have the results been disseminated to communities of interest?DeWalt has also been in contact with biological control practitioners and weed scientists from around the world to bring attention to this species. In addition, the technician and graduate student presented a poster at the Society of Nematology annual meeting in August 2017. DeWalt attended the PD meeting in Washington, D.C. at the end of October 2017 and presented a poster on the findings of the project to date. What do you plan to do during the next reporting period to accomplish the goals?In this next reporting period, we will publish the results of the plant phylogeography study, the common garden experiment, and the herbarium work. We will also continue to collect and receive nematodes to genotype them and inoculate plants of different genotypes to try to achieve objectives #1, #2, and #3. We will sequence the nematodes isolated from different host species and locations for the phylogeography study of the nematode. We will also continue to examine the structure of galls collected in the field from different locations and host plant species to characterize them. We are also planning experiments to sequence the microbiome of galled tissue to compare to non-galled tissue to see if we may be missing one of the partner species in the galls. We have successfully extracted DNA from galls and amplified DNA for the 16S region of bacteria. We are also now conducting an experiment in Trinidad in the field and in the greenhouse with inoculum and plants from Trinidad. If galls develop there, it is possible that the conditions in the greenhouse at Clemson are just not ideal for addressing objectives #2 and #3. We would consider moving our experiments to Trinidad in this case. We are also conducting another experiment in April in the Clemson greenhouse with inoculum from Trinidad on small seedlings of C. hirta from a variety of locations. Although not a major objective of the project, we have been exploring methods to characterize the gall structure to understand better the nematode biology. We have been examining sections of galls made on different host plant species and in different locations. We have also taken pictures of the galls using a stereoscope as well as a camera setup in the Arthropod Museum. Recently, we were able to see live nematodes crawling on the outside of a gall using the stereoscope. We have also isolated some bacteria and fungi from galls from Trinidad by growing them in media. We extracted the DNA and amplified it using the primers ITS1F/ITS4 and 515F/806R, for fungi and bacteria, respectively. We got good amplification for the fungi, but not for the bacteria. The fungal samples will be sequenced.

IMPACT: 2017/01/15 TO 2018/01/14
What was accomplished under these goals? We have identified morphological and genetic variation within the native and introduced ranges of the weed Clidemia hirta, which is an aggressive invader across Hawaii, American Samoa, numerous other islands in the Pacific and Indian Oceans, and continental areas of Africa, Asia, and Australia. From this morphological and genetic variation, we have discovered that the weed in most of its introduced range must have originated from southeastern Brazil. The origin of the weed in Singapore and Indonesia is still in question because it morphologically matches several native genotypes. We are currently examining whether the nematode that creates galls on the aboveground parts of the plant can be used as an effective biological control agent to control this weed in the forests and agricultural lands it invades or whether it is too difficult to propagate. Our major accomplishments to date for objective #1 (to quantify distribution of genetic variation in the host plant species across its native range) have been the following. We conducted a common garden experiment in a greenhouse at Clemson from November 2016 to December 2017 using seeds of Clidemia hirta seed collected from five parts of the invasive range (Singapore, Hawaii, Palau, Pohnpei, La Reunion) and six parts of the native range (Puerto Rico, Dominica, Costa Rica, Brazil, Trinidad, French Guiana). We measured the height and total stem length of these plants at various time points to examine growth rates. We also harvested leaves at different time points to count the number of trichomes, conduct leaf shape analyses, and measure the specific leaf area (a measure of thinness or thickness of the leaves). These data are currently being analyzed to determine the morphological and physiological differences among the genotypes. However, we are able to tell just by looking at a plant whether it is the "Hawaii genotype", which is the predominant genotype in the introduced range and appears closest to plants from southeastern Brazil. Over the past three years, we have also genotyped plants from all of these locations and more using 9 microsatellite loci. We have found that most plants from the introduced range are the same multi-locus genotype that we call the "Hawaii genotype." We found that plants in Singapore and Indonesia are of a different introduced genotype, and morphologically they resemble plants from Puerto Rico, Dominica, Costa Rica, and Trinidad. There are several multi-locus genotypes in each of those parts of the native range. We are also finalizing these analyses. In addition, we examined morphological variation in C. hirta from herbarium specimens collected from across the native range and conducted leaf shape and trichome counts on leaves from these accessions. Although we are still analyzing the data from these three projects, the preliminary results all point to southeastern Brazil being the origin of the plants in Hawaii, Palau, Pohnpei, and La Reunion. We plan to combine this herbarium study, the molecular work, and the results of the common garden study in a manuscript to be written in Summer 2018. Our major accomplishments to date for the other part of objective #1 (to quantify distribution of genetic variation in the galling nematode, which is a potential biocontrol agent, across its native range) have been the following. We collected galls from different species of Melastomataceae and from different locations in Costa Rica (July 2016), Trinidad (June 2016, 2017), and Brazil (2017). We have 20 "populations" from Costa Rica, 32 from Trinidad, and 17 populations from Brazil. A population means that the nematode was collected from a different location within the country and/or different host plant. We have tested for amplification and variation in several genes: ITS, COI, COII, COIII, Cytochrome oxidase B, 12S, 16S, and 18S, and NADH dehydrogenase subunits 1-5 except 3. We will test the IGS region as well. For each of these regions, we have tested several primer pairs that have been used in other nematodes. We were only able to amplify ITS, COI, 12S, 18S, and 12S. The ITS and 18S region was not variable among "populations", but we found some genetic variation among samples from different countries in the COI region. We are waiting to see whether 12S is variable. We also tried whole genome amplification to determine whether we could get enough DNA from a single nematode to do Genotyping by Sequencing, but we were unable to extract more than 0.35 ng, which is not sufficient according to the University of Minnesota Genomics Center. We have been having the most difficulty with our attempts to address objectives #2 and #3 (to assess whether there are differences in pathogenicity and virulence of the different nematode genotypes on different host genotypes, and to measure the benefit in terms of greater pathogenicity gained from optimal matching of plant and nematode genotypes). We have been unable to get galls to develop consistently and to a stage similar to that found in the native range. We therefore have not been able to raise nematodes to address these objectives. We have collected galls, isolated nematodes, and applied inoculum to different genotypes and different sizes of plants in several experiments over the past two years. We have tried to mimic the warm, humid conditions where we find the most galls in the native range of C. hirta by using a misting system in a heated and well-functioning greenhouse at Clemson University. This type of system had been used in Costa Rica and Brazil with Miconia species (a close relative of Clidemia). In July 2016, inoculum with 3000 nematodes from Costa Rica was placed on each of 12 C. hirta plants from Costa Rica and 8 plants from Hawaii. Galls were observed on the Hawaiian plants about 60 days after the inoculation. The galls did not develop very far, the plants still seemed healthy, and we could not find any nematodes on the plants 120 days after inoculation. In March 2017, we inoculated small seedlings of C. hirta from Trinidad and Hawaii. The number of nematodes varied according to the source plant (100 to 4889 nematodes/application). This time all the plants showed some gall development, beginning after only 20 days. The galling appeared more severe (more leaves affected, more galls on each leaf) on the Hawaiian plants than the Trinidad plants. After three months, the galls once again stopped developing, and we were unable to find any nematodes in the galls 120 days after inoculation. In June 2017, we conducted three experiments using nematodes we isolated from galls we had collected in Trinidad. We applied 4000 nematodes per plant to different size plants (small seedlings, medium-sized, and large-sized plants) and genotypes (Hawaii, Costa Rica, Dominica, Puerto Rico, Singapore) or using different sources of nematodes (different Melastomataceae species). We also tried using different inoculum levels on the same plant with 4000, 7000, and 10,000 nematodes added to different locations on 4 different medium-sized plants from Costa Rica. None of the plants in three experiments developed symptoms. In December 2017, we once again inoculated big plants and very small plants (all different genotypes) with 3000 nematodes/plant using inoculum from Trinidad. We tried attaching some galls to the plants. We found one gall about 60 days after inoculation, but it did not develop and no nematodes were found within it.

PUBLICATIONS: 2017/01/15 TO 2018/01/14
No publications reported this period.

PROGRESS: 2016/01/15 TO 2017/01/14
Target Audience:We sponsored research by one female graduate student, made contact with weed scientists in several areas where Clidemia hirta is invasive, and worked with plant ecologists and pathologists throughout the native range of C. hirta. Changes/Problems:We had no major changes in approach except that it is taking longer than expected to determine ideal rearing conditions for the nematode. What opportunities for training and professional development has the project provided?We continue to employ a full-time technician, and this person is taking care of the plants. She is planning to start graduate school in Fall 2017. We also supported the honors thesis research of a female undergraduate student majoring in Genetics. This past year, we hired a PhD graduate student from Brazil to work on the project in August 2017. Her contacts with nematologists and plant pathologists in Brazil will prove very useful in meeting the aims of this project. How have the results been disseminated to communities of interest?DeWalt presented some of this work at the Association for Tropical Biology and Conservation conference in Montpellier, France in June 2016. In addition, she gave a symposium presentation on the role of pathogens and herbivores on plant invasions at the Ecological Society of America meetings in Ft. Lauderdale, Florida in August 2016. She has also been in contact with biological control practitioners and weed scientists from around the world to bring attention to this species. What do you plan to do during the next reporting period to accomplish the goals?In this next reporting period, we will continue the common-garden experiment in the greenhouse, and incorporate the results in the manuscript being prepared to examine the phylogeography of Clidemia hirta. We will also continue to collect and receive nematodes to genotype them and inoculate plants of different genotypes to achieve supporting objectives #1, #2, and #3. We will continue different primer combinations to genotype the nematode isolated from different host species and locations for the phylogeography study of the nematode. We will work with a lab in Brazil to optimize nematode extraction, inoculation, and propagation.

IMPACT: 2016/01/15 TO 2017/01/14
What was accomplished under these goals? This year we have accomplished many of the goals we set out for the second year of this grant, which were to do the following: plant seeds in the greenhouse for rearing nematodes; genotype plant material; publish plant phylogeography study; collect and import nematodes from 8 native locations; genotype nematode material; isolate nematodes and apply inoculum to rearing plants. For 1-3: We planted seeds of Clidemia hirta seed from five parts of the invasive range (Singapore, Hawaii, Palau, Pohnpei, La Reunion) and six parts of the native range (Puerto Rico, Dominica, Costa Rica, Brazil, Trinidad, French Guiana). Leaves from all of these sites have been genotyped, and we additionally received leaves from other locations of the invasive range that we are in midst of genotyping (e.g. Papua New Guinea, Java). As we have continued to receive leaf samples, we have not yet published the phylogeographic study examining the amount of genetic variation in the native range and from where in the native range the introduced range genotypes may stem. In addition in year 1, an undergraduate student conducted a side project to look at morphological variation in C. hirta from herbarium specimens. We plan to combine this herbarium study, the molecular work, and the results of the currently running common garden study in a manuscript to be written in Summer 2017. For 4-6: We collected galls on different melastome species and locations in Trinidad (June 2016) and Costa Rica (July 2016) this year and sent them back to Clemson intact. The graduate student employed on the grant has been trying different primer combinations to genotype the nematodes for the nematode phylogeography study. We isolated the nematodes and applied inoculum to cuttings or plants grown from seed. We were unsuccessful getting the nematodes from Trinidad to infect plants grown from Puerto Rican and Costa Rican plants, but it was likely because the plants were not growing in high enough humidity. We did get symptoms (small galls) to develop on Hawaiian cuttings, but not Costa Rican plants grown from seeds, about 2 months after applying inoculum isolated from Costa Rican galls. Strangely, the galls stopped developing soon after they were started, and no nematode reproduction seems to have occurred in or near these galls. We now have an effective misting system in the greenhouse that should keep the humidity high enough for nematodes to establish, and we are testing different sizes of plants to determine ideal rearing conditions. We are continuing experiments with nematodes from Trinidad and small plants grown from seed from Trinidad. We are conducting histology on galls collected in the field and greenhouse to determine the shape and nature of the galls. We are also planning experiments to sequence the microbiome of galled tissue to compare to non-galled tissue to see if we may be missing one of the partner species in the galls. We are also collaborating with a lab in Brazil who is trying to determine how best to propagate the nematodes for use in experiments.

PUBLICATIONS: 2016/01/15 TO 2017/01/14
No publications reported this period.

PROGRESS: 2015/01/15 TO 2016/01/14
Target Audience:We sponsored research by one female undergraduate student and made contact with weed scientists in several areas where Clidemia hirta is invasive. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We hired a full-time technician, and this person is taking care of the plants as well as genotyping the nematodes and plants we have collected. She is planning to attend graduate school in the near future. We have also supported the honors thesis research of an undergraduate student majoring in Genetics. How have the results been disseminated to communities of interest?DeWalt presented some of this work at the Association for Tropical Biology and Conservation conference in Hawaii in July 2015 and will present more of it at the same conference in Montpellier, France in June 2016. In addition, she will give a symposium presentation on the role of pathogens and herbivores on plant invasions at the Ecological Society of America meetings in Ft. Lauderdale, Florida in August 2016. She has also been in contact with biological control practitioners and weed scientists from around the world to bring attention to this species. What do you plan to do during the next reporting period to accomplish the goals?In this next reporting period, we will begin the common-garden experiments and inoculations of nematodes to achieve objectives #2 and #3. We will hire a graduate student to conduct the nematology work to begin Fall 2016.

IMPACT: 2015/01/15 TO 2016/01/14
What was accomplished under these goals? This year we have accomplished the goals we set out for the first year of this grant, which were to collect seeds from the native and invasive ranges. We collected Clidemia hirta seed or had it sent to us from parts of the invasive range (Singapore, Hawaii, Palau, Pohnpei) and native range (Puerto Rico, Dominica, Costa Rica, Brazil). Additionally, we are waiting on seed to be sent to us from Trinidad, French Guiana, and Guyana. We collected nematodes from Costa Rica, Brazil, and Guyana, but only the ones from Brazil arrived alive due to delays in shipping the ones from Costa Rica and Guyana. We were unsuccessful getting the nematodes from Brazil to infect some plants grown from cuttings sent from Hawaii, but this was likely because the humidity was too low in the environmental chamber. We now have a system in place to get the nematodes to live. We planted seed from the native and invasive range and are waiting for the plants to grow. They should be large enough to try infecting with nematodes by June. We will collect nematodes from Guyana, Brazil, and Costa Rica again this summer, which is on schedule. We have genotyped many more Clidemia hirta from around the native range. We are conducting a small side project to look at morphological variation in C. hirta, and an undergraduate student is measuring trichome density and leaf shape on herbarium specimens we have on loan from the New York Botanical Garden (NYGB). We also have permission from NYBG to genotype their samples, and therefore we are adding to our phylogeography study with 90 more specimens.

PUBLICATIONS: 2015/01/15 TO 2016/01/14
No publications reported this period.