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Enhancing cropping intensity is the most effective and significant method to improve regional crop production and ensure food security. However, our understanding of the impacts of reduced cropping intensity on soil quality and ecosystem multifunctionality (EMF) with soil depth remains incomplete in tropical regions. Here, we performed a 4-year field experiment to estimate the impacts of cropping intensity (continuous cropping, winter fallow, and annual fallow) on soil quality and EMF depending on soil depths. We found that reduced cropping intensity improved soil quality at the topsoil (0–10 cm), while it had no significant influences on soil carbon (C) and nitrogen (N) storage, as well as soil EMF at 0–40 cm. Soil microbes were limited by C and P but not co-limited by N in all three cropping systems. Reduced cropping intensity exacerbated microbial C limitation at 0–10 cm due to reduced additional C resources (i.e., rice straw and manure) input. Redundancy analysis and Pearson correlation showed that soil N significantly affected the C-, N-, and P-acquisition enzyme activities, and correlated positively with soil organic C, microbial biomass C, and available P. In conclusion, short-term reduction in cropping intensity improves topsoil quality but not soil EMF under paddy-upland rotations in the tropical region.

The rhizosphere and detritusphere are hotspots of soil enzyme-mediated microbial processes, but little is known about their spatio-temporal distribution and interactions in situ, especially after straw incorporation. To answer this question, we planted maize in rhizoboxes with three wheat straw localizations: a) straw incorporated in the upper 2 cm of soil (straw localized); b) straw mixed with all the soil (straw homogenized); and 3) without straw (control). Zymography was used to investigate the spatio-temporal distribution of soil C- and N- acquiring enzyme activities in the rhizosphere (living roots) and detritusphere (straw induced), which depends on straw localization (localized vs. homogenized) and time (7 vs. 15 days after planting). The hotspot area of enzyme activities was similar in the detritusphere compared to the rhizosphere at the early stage (day 7), suggesting the importance of detritusphere in driving C and N cycling. Compared with homogenized straw, higher C- and N-acquiring enzyme activities in detritusphere were observed in the treatment with localized straw, which indicates that straw localization had a marked impact on the enzyme activity in the detritusphere. Although enzyme activities in rhizosphere and detritusphere decreased with time from straw application, straw incorporation increased the enzyme activities in the rhizosphere (especially with localized straw) compared to control at the late stage (day 15). Furthermore, homogenized straw greatly reduced plant height (−36 %), leaf area (−49 %), SPAD (−49 %), and shoot weight (−53 %) than control at the late stage, but localized straw had little impact on plant performance. In conclusion, straw incorporation forms another soil enzyme activity hotspot (detritusphere) and promotes enzyme activities in the rhizosphere and soil profile. Collectively, homogenized straw incorporation intensified nutrient competition between microorganisms and roots and restricted plant performance.

Nitrous oxide (N2O) emissions from agroecosystems are a major contributor to global warming and stratospheric ozone depletion. However, knowledge concerning the hotspots and hot moments of soil N2O emissions with manure application and irrigation, as well as the underlying mechanisms remain incomplete. Here, a 3-year field experiment was conducted with the combination of fertilization (no fertilizer, F0; 100% chemical fertilizer N, Fc; 50% chemical N + 50% manure N, Fc + m; and 100% manure N, Fm) and irrigation (with irrigation, W1; and without irrigation, W0; at wheat jointing stage) for winter wheat – summer maize cropping system in the North China Plain. Results showed that irrigation did not affect annual N2O emissions of the wheat-maize system. Manure application (Fc + m and Fm) reduced annual N2O emissions by 25–51% compared with Fc, which mainly occurred during 2 weeks after fertilization combined with irrigation (or heavy rainfall). In particular, Fc + m reduced the cumulative N2O emissions during 2 weeks after winter wheat sowing and summer maize top dressing by 0.28 and 0.11 kg ha−1, respectively, compared with Fc. Meanwhile, Fm maintained the grain N yield and Fc + m increased grain N yield by 8% compared with Fc under W1. Overall, Fm maintained the annual grain N yield and lower N2O emissions compared to Fc under W0, and Fc + m increased the annual grain N yield and maintained N2O emissions compared with Fc under W1, respectively. Our results provide scientific support for using manure to minimize N2O emissions while maintaining crop N yield under optimal irrigation to support the green transition in agricultural production.

Purpose: Intercropping is an important agricultural management that has been applied worldwide. Although intercropping improves soil nutrients and crop productivity, its effects on the microbial-mediated belowground processes and main drivers remain unclear. Methods: We performed the same field study at two sites (Site1, Youyu; Site2, Zhangbei) by growing soybean and oat in monoculture and intercropping to investigate their effects on rhizosphere soil properties, enzyme stoichiometry, and soil ecosystem multifunctionality (EMF). Results: Intercropping increased available phosphorus (Avail-P) by 87% and 16% for oat and soybean compared to the corresponding monoculture in site1, respectively. We also found that intercropping increased the C-acquiring and N-acquiring enzyme activities by 18%-48% in site1. Moreover, intercropping enhanced soil EMF and alleviated microbial P limitations for both oat and soybean compared to the corresponding monoculture in site1. However, all observed parameters were not affected by intercropping in site2, which may be due to the lower Avail-P, mineral nitrogen (Nmin), and precipitation in site2 compared to site1. Moreover, the soil EMF was strongly positively correlated with soil Nmin, Avail-P, air temperature, and precipitation. Conclusion: Therefore, intercropping improves soil ecosystem multifunctionality by increasing available nutrients, which are regulated by regional factors.

Subsurface organic ameliorant plus polyethylene mulching is a novel strategy for saline soil improvement and utilization in China. However, little is known about how this strategy impacts soil organic carbon (SOC) both directly (carbon input) and indirectly (soil aggregates protection and microbial regulation). Therefore, a field experiment was arranged and sampled after 3 years, including four treatments, i.e., no ameliorant with and without polyethylene mulching, and subsurface (10–30 cm soil depth) organic ameliorant with and without polyethylene mulching. Subsurface organic ameliorant raised the SOC content in the 0–40 cm by 70% and 90% with and without mulching, respectively. Subsurface organic ameliorant converted the major aggregation size from

Mineral fertilizers and livestock manure are major sources of nitrous oxide (N2O) emissions from agricultural soil. Meta-analyses consistently show that nitrification inhibitors reduce N2O emissions, and this study was established to evaluate the efficacy of three selected compounds, DMPP, nitrapyrin and Piadin, to reduce N2O emissions from spring-applied mineral fertilizers (NS 26–13 or UAN) and pig slurry. An N source either alone or combined with an inhibitor were established in 2020 and 2021. In 2020 these treatments were applied in late April before seeding of spring barley, and in 2021 the same treatments were applied in early April to winter wheat under conventional tillage (as in 2020) and no-till management, respectively. Nitrous oxide fluxes and soil mineral N dynamics were monitored during spring only. The range of N2O emissions in the spring barley experiment was typical for this site, bt much higher with pig slurry compared with mineral fertilizers. The winter wheat experiment the following year showed generally low N2O emissions. The effects of nitrification inhibitors, across all treatments, varied between 0 and 90% reduction of N2O emissions. The variable emission levels and treatment effects were explained with reference to climate (temperature, rainfall), distribution of fertilizers, and crop growth, and a need for comparative studies to resolve interacting effects was identified. Overall, the results were consistent with nitrification inhibitors being more effective at reducing N2O emissions from crops where mineral N uptake is delayed relative to the time of fertilization, from mineral N fertilizers in wet or compacted soil, and from organic fertilizers at moderate or even dry soil conditions.

Purpose Soil bacteria and fungi play critical roles in mediating soil nutrient cycling. Exploring the effect of fertilization on soil microbial communities is of great importance to comprehend the sustainability of agricultural ecosystem. Methods In this study, responses of the bacterial and fungal communities in the wheat rhizosphere and bulk soils to four fertilization regimes: no fertilizer (CK), 100% chemical NPK fertilizers (NPK), 50% chemical NPK fertilizers + 50% manure (NPKM), and 100% manure (OM) were investigated. Results Chemical fertilization significantly decreased the bacterial alpha-diversity, while manure fertilization maintained the bacterial diversity in both rhizosphere and bulk soils. Fertilization increased the relative abundance of the bacterial orders Flavobacteriales and Xanthomonadales by 46.0-61.6% and 36.3-59.4% compared with CK in the rhizosphere soils. Application of both chemical fertilizers and manure enhanced the bacterial network complexity and stability. Chemical fertilizer addition significantly weakened the fungal network complexity, while manure fertilization maintained the fungal network complexity and connectivity. Proteobacteria and Bacteroidetes were the two prominent keystone taxa of bacterial networks in CK and NPK, while Actinobacteria was the prominent keystone taxa in NPKM and OM. In addition, Myrothecium became the keystone taxa of fungal networks induced by manure addition. Conclusions Our results suggest that soil bacteria were more sensitive than fungi to fertilization, and manure amendment was beneficial to increase soil microbial network complexity and stability. These findings provide a comprehensive understanding of how fertilization affects the productivity and sustainability of agricultural ecosystems via soil microbial communities.

Soil organic matter (SOM) decomposition in response to global change represents a critical uncertainty in coupled carbon (C) cycle-climate models. Much of this uncertainty arises from our limited mechanistic knowledge of the effects of organic C input frequency on SOM decomposition. Based on a three-source-partitioning isotopic approach (14C glucose addition to soil continuously labeled by C4 plants over 21 years) and literature review of 86 observations, we assessed the priming of fast- and decadal-SOM decomposition after occasional and frequent (every 12 vs 60 days) labile C input in a grassland soil. Frequent glucose input accelerated SOM decomposition over 200 days, but the occasional input reduced this positive priming by 2.6 times. The positive priming by occasional C input resulted in 139 μg C g−1 soil net C sequestration corresponding to 38% of C input. During the 200 days incubation, the primed fast-cycling C (younger than 21 years) under occasional C addition was 52–94% greater compared with frequent addition. Conversely, the priming of decadal SOM (older than 21 years) by frequent C input was 463 μg C g−1 after the 200 days incubation, which represents 5.3% of the initial decadal-cycling soil C pool. This was 1.5 times higher than by occasional input because of the stronger N mining caused by the continuously high activity of microbial K strategists under frequent C input. Our global scale assessment showed that occasional C input reduces the priming of decadal SOM decomposition by 89–127 Tg per year compared to frequent input and thus, sequestrated an additional 102 Tg C per year. Overall, the vulnerability of decadal SOM to increasing C input frequency thus weakening the C sink of grassland, whilst reduced C input frequency (e.g. by drought) suppresses the SOM priming and reduces the positive feedback of SOM decomposition to global change. This points out the necessity to assess the age of soil C when predicting the consequences of altered soil C input frequency under global change.

Proteins are the main N compounds and are important sources of both C and N for plants and microbes. Triple-labeled dissolved organic N obtained from white clover shoots was separated into five different molecular weight (Mw) size fractions (> 1 kDa). In this study, we investigated the mineralization of and the presence of C and N remaining in soil solution over 14 days across different soil pH values. The evolved CO2 decreased by increasing Mw, suggesting an apparent bottleneck in the decomposition of organic N above Mw 30 kDa. The initial parallel loss of 13C and 15 N from soil was followed by an increase in dissolved 15 N describing a two-phase process of organic N turnover. The first-phase concerns the removal of organic N whereas the second-phase concerns the release of inorganic N. Microbial N and C use efficiency was increased with increasing Mw size suggesting the importance of organic N quality in predicting the impact on soil C and N cycling.

Soil aggregates not only store carbon (C) and nitrogen (N) but hold a critical role in determining the nutrients supply, crop productivity, and climate change mitigation. However, the impact of cropping system and N fertilization on aggregate-associated C and N in both topsoil and subsoil remains unclear. Here, we assessed the effect of cropping systems (wheat–soybean vs. wheat–maize cropping systems) and N fertilization rates (0 N; medium N, 120 kg N ha−1; high N, 240 kg N ha−1) on soil water-stable aggregates distribution, as well as aggregate-associated C and N based on a field study in North China Plain. Our study suggests that the variations of soil organic carbon (SOC) and total nitrogen (TN) stocks were more affected by N fertilization than short-term cropping systems. In the wheat–soybean system, medium N increased the SOC stock by 19.18% and 15.73% as compared to high N in the topsoil and subsoil, respectively. Additionally, medium N resulted in 6.59–18.11% higher TN stock in the topsoil for both wheat–soybean and wheat–maize cropping systems as compared to 0 N and high N. Notably, the water-stable macroaggregates (> 0.25 mm) in the topsoil occupied more than 70% of the soil, which increased under medium N in the wheat–soybean cropping system. In conclusion, medium N fertilization combined with a legume-based cropping could be used to improve SOC stock, promote soil aggregation, and enhance aggregate-associated C.

The cycling of especially large size organic nitrogen (N) from plants into stable microbial derived soil organic carbon (C) and N pools is understudied, in spite of organic N composing 90% of soil N and the intimate link between organic N and soil C stabilization. We investigated the fate of peptide-size and protein-size organic N fractions in soils from two long-term field experiments markedly differing in conditions for microorganisms. We combined amino acid stable isotope probing (AA-SIP) fingerprinting with PLFA-SIP to trace organic N into the soil microbial biomass. Contrary to the present paradigm, we found for both soils that greater molecular size did not protect against decomposition of these compounds neither did protection via strong sorption to the soil mineral phase. Instead, we found strong evidence that gram-positive bacteria are the key actors in the decomposition of protein-sized nitrogen compounds and that amino acids bound in large organic nitrogen compounds directly contribute to the build-up of bacterial tissue. We conclude that when large organic nitrogen compounds are dissolved, turnover occurs rapidly, irrespective of molecular size, and the bacterial incorporation of these rapid cycling compounds makes an important contribution to soil organic matter formation.

Background: Soil organic matter (SOM) formed under legume-based grasslands may serve as an important source of nitrogen (N) for subsequent maize growth. Aim: We aimed at studying effects of fertilizer type (organic and mineral N and phosphorus (P) fertilizers) on N release from recently formed SOM and N utilization by plants. Methods: The effects of mineral and organic (meat and bone meal) N and P fertilization on SOM turnover were examined under maize (Zea mays L.) on a sand loamy typic hapludult in an outdoor pot experiment over 55 days. The plant uptake of N derived from recently formed SOM was investigated by dual labeling this SOM pool with 15N and 13C. Results: Organic and mineral fertilized maize acquired similar amounts of N derived from recently formed SOM, which was twice as high as for unfertilized plants. Rhizosphere soil with organic fertilizer had higher 13C loss than unplanted soil, whereas no extra 13C was lost under mineral fertilization. On the contrary, a lower 13C loss from planted compared to unplanted soil was observed in unfertilized soil. Conclusions: Both mineral and organic fertilizers increased plant growth and N uptake, whereas only organic fertilizers accelerated SOM turnover in the rhizosphere as compared to bulk soil. Thus, this study implies that the fertilizer types differently affected the mechanisms controlling the utilization of N bound in recently formed SOM.

Estimates of nitrogen (N) losses from living plants (phyllo- and rhizodeposition) are needed to improve the sustainability of the present agricultural cropping systems. These estimates are hard to achieve under field conditions among others due to a lack of suitable methods to study these N flows. A novel vertical split-root with 15N placement in deeper soils layers is suggested here as a means to improve the present tracer based methods for phyllo- and rhizodeposition estimation. We found enrichment of soil above the point of tracer injection (at 290 cm) with rhizosphere having higher enrichment than bulk soil underlining that 15N was derived from roots. The relative net N rhizodeposition was highest for lucerne and decreased with depth for all three plant species investigated (lucerne, kernza®, rosinweed). The quantity of N lost via rhizodeposition amounted to 10–13 kg N ha−1 in the topsoil, but the majority of the N rhizodeposition was found below (25–250 cm) showing the importance of including deeper soil layers in such studies.

Cover crops (CC) increase soil fertility via recycling of nutrients such as nitrogen (N) and buildup of soil organic matter, whereby CC contributes to climate change mitigation. The carbon (C) input to soil occurs via the input of plant material (shoot and root) and via phyllo- and rhizodeposition. However, the quantity of phyllo- and rhizodeposition from CC is largely unexamined, although this component potentially has a large contribution to microbial stabilization of C. Isotopic labeling with 13C to trace C flows and 15N to determine N2-fixation was conducted to test the short-term effect of the inclusion of winter vetch as a legume species to a CC mixture. The CC mixtures were established in a long-term experiment (1997–2019) with varying use of manure and CC cropping to test the effect of soil fertility on the behavior of the newly established CC mixtures. A significant reduction in the relative net phyllo- and rhizodeposition was observed with higher soil fertility. Interestingly, the quantity of net phyllo- and rhizodeposition was also reduced with higher soil fertility due to the lower relative phyllo- and rhizodeposition, despite a significantly higher CC biomass. Importantly, adjusting for unrecovered root fragments more than halved the calculated phyllo- and rhizodeposition, indicating that many published estimates are substantially overestimated. The use of CC during autumn provides an important window for C storage with both the input of plant biomass and belowground C investment being drivers of C storage.

The growth of deep-rooted crops has the potential to increase carbon deposition within deep subsoil layers, thereby contributing to climate change mitigation. Yet, the mechanisms of labile carbon stabilization in deep subsoils remain uncertain. Here we studied the effect of nitrogen, phosphorus and sulphur limitations on the microbial mineralization of added glucose and an artificial root exudate mixture (both spiked with 14C-glucose) in intact soil samples from a deep subsoil (5–6 m) over a 10-week incubation period, where the temporal respiration of CO2 and 14CO2 were quantified. We found that mineralization was co-limited by nitrogen and phosphorus, but the cumulative amount of CO2 emitted was significantly larger from artificial root exudates than glucose, suggesting that the nitrogen carried in amino acids in root exudates (here, L-arginine) was sufficient to overcome the nitrogen limitation in this subsoil. Highlights: Labile C substrates and nutrients from deep-rooted crops may affect the potential for C sequestration in subsoil. Deep subsoil was incubated with glucose and artificial root exudates amended with N, P, and S. C mineralization was co-limited by N and P, but less so in soil amended with glucose. Amino acid derived-N in exudates from deep-rooted crops may alleviate the N limitation in deep subsoils.

Quantification of legume nitrogen (N) rhizodeposition (N derived from roots) and its fate in agroecosystems is crucial for managing soil fertility, land productivity, and agriculture sustainability. In contrast to forage legumes, the N rhizodeposition by grain legumes is nearly unknown. Therefore, N rhizodeposition of four grain legumes and its transfer to subsequent wheat crops was quantified using the(15)N stem labeling method under field conditions. The N rhizodeposition of the grain legumes: peanut, soybean, mungbean, and adzuki bean amounted to 25, 51, 20, and 63 kg N ha(-1), respectively. N rhizodeposition was not affected by fertilization, and it was 53-257% more accumulated in topsoil (0-20 cm) than that in subsoil (20-40 cm). However, N rhizodeposition per unit of root biomass in subsoil was 3.5-times as much as that in topsoil (p

Despite the importance of subsoil carbon (C) deposition by deep-rooted crops in mitigating climate change and maintaining soil health, the quantification of root C input and its microbial utilization and stabilization below 1 m depth remains unexplored. We studied C input by three perennial deep-rooted plants (lucerne, kernza, and rosinweed) grown in a unique 4-m deep RootTower facility. 13C multiple pulse labeling was applied to trace C flows in roots, rhizodeposition, and soil as well as 13C incorporation into microbial groups by phospholipid fatty acids and the long-term stabilization of microbial residues by amino sugars. The ratio of rhizodeposited 13C in the PLFA and amino sugar pools was used to compare the relative microbial stability of rhizodeposited C across depths and plant species. Belowground C allocation between roots, rhizodeposits, and living and dead microorganisms indicated depth dependent plant investment. Rhizodeposition as a fraction of the total belowground C input declined from the topsoil (0–25 cm) to the deepest layer (360 cm), i.e., from 35%, 45%, and 36%–8.0%, 2.5%, and 2.7% for lucerne, kernza, and rosinweed, respectively, where lucerne had greater C input than the other species between 340 and 360 cm. The relative microbial stabilization of rhizodeposits in the subsoil across all species showed a dominance of recently assimilated C in microbial necromass, thus indicating a higher microbial stabilization of rhizodeposited C with depth. In conclusion, we traced photosynthates down to 3.6 m soil depth and showed that even relatively small C amounts allocated to deep soil layers will become microbially stabilized. Thus, deep-rooted crops, in particular lucerne are important for stabilization and storage of C over long time scales in deep soil.

Methane (CH4) emission is the consequence of CH4 production and consumption performed by methanogens and methanotrophs, respectively. Fertilization is an important factor that regulates the behavior of methanogens and methanotrophs; however, the effect of manure and rice straw addition combined with inorganic fertilizers on these communities is not well understood. This study aimed to explore how manure and rice straw amendments together with inorganic fertilizers influenced the methanogenic and methanotrophic communities in a 31-year fertilized rice paddy. Manure amendment significantly increased the abundance of mcrA and pmoA genes by 61.2% and 63.3% compared with the unfertilized control, whereas inorganic NPK fertilization alone or rice straw addition did not affect their abundances. Manure and rice straw amendments greatly decreased the Shannon index and ACE index of the methanogenic communities, whereas inorganic NPK fertilization alone increased the ACE index of the methanotrophic communities compared with the unfertilized control. Methanosarcinaceae and Methylococcaceae dominated at the family level, representing 23.1-35.0% and 48.7-67.2% of the total reads, for the methanogenic and methanotrophic communities, respectively. Application of manure together with inorganic fertilizers suppressed the Methanocellales methanogens and the type I methanotrophs (Methylococcus and Methylobacter). Fertilization greatly altered the community structure of methanogens and methanotrophs, and manure addition had more apparent effects than rice straw. Moreover, total nitrogen, soil organic carbon, available phosphorus, and available potassium correlated significantly to the abundance, composition, and community structure of methanogens and methanotrophs. In conclusion, our study revealed that long-term manure amendment in combination with inorganic fertilizers significantly increased the abundance of methanogens and methanotrophs, but suppressed the type I methanotrophs in rice paddies.

Ensign wasps (Hymenoptera: Evaniidae) develop as predators of cockroach eggs (Blattodea), have a wide distribution and exhibit numerous interesting biological phenomena. The taxonomy of this lineage has been the subject of several recent, intensive efforts, but the lineage lacked a robust phylogeny. In this paper we present a new phylogeny, based on increased taxonomic sampling and data from six molecular markers (mitochondrial 16S and COI, and nuclear markers 28S, RPS23, CAD, and AM2), the latter used for the first time in phylogenetic reconstruction. Our intent is to provide a robust phylogeny that will stabilize and facilitate revision of the higher-level classification. We also show the continued utility of molecular motifs, especially the presence of an intron in the RPS23 fragments of certain taxa, to diagnose evaniid clades and assist with taxonomic classification. Furthermore, we estimate divergence times among evaniid lineages for the first time, using multiple fossil calibrations. Evaniidae radiated primarily in the Early Cretaceous (134.1-141.1 Mya), with and most extant genera diverging near the K-T boundary. The estimated phylogeny reveals a more robust topology than previous efforts, with the recovery of more monophyletic taxa and better higher-level resolution. The results facilitate a change in ensign wasp taxonomy, with Parevania, syn. nov., and Papatuka, syn. nov. becoming junior synonyms of Zeuxevania, and Acanthinevania, syn. nov. being designated as junior synonym of Szepligetella. We transfer 30 species to Zeuxevania, either reestablishing past combinations or as new combinations. We also transfer 20 species from Acanthinevania to Szepligetella as new combinations.

The invasive eucalyptus tortoise beetle, Paropsis charybdis, defoliates plantations of Eucalyptus nitens in New Zealand. Recent efforts to identify host specific biological control agents (parasitoids) from Tasmania, Australia, have focused on the larval parasitoid wasp, Eadya paropsidis (Braconidae), first described in 1978. In Tasmania, Eadya has been reared from Paropsisterna agricola (genus abbreviated Pst.), a smaller paropsine that feeds as a larva on juvenile rather than adult foliage of Eucalyptus nitens. To determine which of the many paropsine beetle hosts native to Tasmania are utilized by E. paropsidis, and to rule out the presence of cryptic species, a molecular phylogenetic approach was combined with host data from rearing experiments from multiple locations across six years. Sampling included 188 wasps and 94 beetles for molecular data alone. Two mitochondrial genes (COI and Cytb) and one nuclear gene (28S) were analyzed to assess the species limits in the parasitoid wasps. The mitochondrial genes were congruent in delimiting four separate phylogenetic species, all supported by morphological examinations of Eadya specimens collected throughout Tasmania. Eadya paropsidis was true to the type description, and was almost exclusively associated with P. tasmanica. A new cryptic species similar to E. paropsidis, Eadya sp. 3, was readily reared from Pst. agricola and P. charybdis from all sites and all years. Eadya sp. 3 represents the best candidate for biological control of P. charybdis and was determined as the species undergoing host range testing in New Zealand for its potential as a biological control agent. Another new species, Eadya sp. 1, was morphologically distinctive and attacked multiple hosts. The most common host was Pst. variicollis, but was also reared from Pst. nobilitata and Pst. selmani. Eadya sp. 1 may have potential for control against Pst. variicollis, a new incursion in New Zealand, and possibly Pst. selmani in Ireland. Our molecular data suggests that Pst. variicollis is in need of taxonomic revision and the geographic source of the beetle in New Zealand may not be Tasmania. Eadya sp. 2 was rarely collected and attacked P. aegrota elliotti and P. charybdis. Most species of Eadya present in Tasmania are not host specific to one beetle species alone, but demonstrate some host plasticity across the genera Paropsisterna and Paropsis. This study is an excellent example of collaborative phylogenetic and biological control research prior to the release of prospective biological control agents, and has important implications for the Eucalyptus industry worldwide.