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Red radish (Raphanus sativus) is highly susceptible to the soil-borne fungus Rhizoctonia solani, which can cause severe crop losses. In a glasshouse experiment, untreated seeds of radish cvs. French Breakfast and Red Round were grown in potting mix where R. solani inoculated wheat-bran was added at rates of 0.25, 0.5 and 1.0 g per 100 g potting mix. Seedling emergence was reduced by one third and two thirds respectively by the two higher inoculum rates, and final plant numbers were ca. 20%, 50% and 80% less than in the uninoculated control. The ability of Trichoderma spp. to increase radish yields by limiting the damage caused by R. solani has long been known but has not been evaluated in New Zealand. Inoculum of each of four Trichoderma spp. isolates LU132 (T. atroviride), LU785 (T. hamatum), LU1437 (T. harzianum) and LU1358 (T. polysporum) was prepared in sterile wheat-bran and 0.5 g wheat-bran was added per 100 g potting mix. In a second glasshouse experiment, R. solani (0.25 g inoculated wheat-bran) was added per 100 g potting mix before untreated seeds of both radish cultivars were sown. Potting mix without either R. solani or Trichoderma served as the control. Maximum seedling emergence did not differ among the treatments for cv. French Breakfast, but was increased by the presence of either isolate LU132 (T. atroviride) or LU1358 (T. polysporum) for cv. Red Round. The presence of isolate LU1347 (T. harzianum) in the potting mix significantly increased plant survival in both cultivars. Each of the four Trichoderma isolates reduced the percentage of diseased plants with isolate LU132 (T. atroviride) providing the strongest response. In a third glasshouse experiment, Trichoderma treated seeds, thiram fungicide treated and untreated seeds of both radish cultivars were sown in naturally R. solani infected soil. The same treatments were used in a field trial at a site known to be infected by R. solani. In the third glasshouse experiment, seed treatment with Trichoderma isolates LU1347 (T. harzianum), LU1358 (T. polysporum) and LU785 (T. hamatum) significantly increased bulb fresh weight in cv. Red Round, but no treatments increased bulb fresh weight in cv. French Breakfast. In the field experiment, bulb yield for the thiram seed treatment did not differ from that of the untreated control. However, seed treatment with isolate LU785 (T. hamatum) increased subsequent bulb yield by 96% for both cultivars, and seed treatment with isolate LU132 (T. atroviride) or isolate LU1358 (T. polysporum) also significantly increased bulb yield (by 85% and 60% respectively) in cv. French Breakfast. A possible explanation for this result was sought by undertaking a fourth glasshouse experiment for radish cv. Red Round only. In this experiment, all four Trichoderma spp. isolates more than doubled bulb yield by producing not only a greater number of bulbs but also larger bulbs than the untreated control. Trichoderma seed coating may provide an alternative to fungicide seed treatment for radish production.

Abstract Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium. Insect pathogenic strains have been characterised in New Zealand, and two isolates, Bl 1821L and Bl 1951, are under development for use in biopesticides. However, growth in culture is sometimes disrupted, affecting mass production. Based on previous work, it was hypothesised that Tectiviridae phages might be implicated. While investigating the cause of the disrupted growth, electron micrographs of crude lysates showed structural components of putative phages including capsid and tail-like structures. Sucrose density gradient purification yielded a putative self-killing protein of ~30 kDa. N-terminal sequencing of the ~30 kDa protein identified matches to a predicted 25 kDa hypothetical and a 31.4 kDa putative encapsulating protein homologs, with the genes encoding each protein adjacent in the genomes. BLASTp analysis of the homologs of 31.4 kDa amino acid sequences shared 98.6% amino acid identity to the Linocin M18 bacteriocin family protein of Brevibacterium sp. JNUCC-42. Bioinformatic tools including AMPA and CellPPD defined that the bactericidal potential originated from a putative encapsulating protein. Antagonistic activity of the ~30 kDa encapsulating protein of Bl 1821L and Bl 1951during growth in broth exhibited bacterial autolytic activity. LIVE/DEAD staining of Bl 1821L cells after treatment with the ~30 kDa encapsulating protein of Bl 1821L substantiated the findings by showing 58.8% cells with the compromised cell membranes as compared to 37.5% cells in the control. Furthermore, antibacterial activity of the identified proteins of Bl 1821L was validated through gene expression in a Gram-positive bacterium Bacillus subtilis WB800N. Key Points • Gene encoding the 31.4 kDa antibacterial Linocin M18 protein was identified • It defined the autocidal activity of Linocin M18 (encapsulating) protein • Identified the possible killing mechanism of the encapsulins

During infection, phages manipulate bacteria to redirect metabolism towards viral proliferation. To counteract phages, some bacteria employ CRISPR-Cas systems that provide adaptive immunity. While CRISPR-Cas mechanisms have been studied extensively, their effects on both the phage and the host during phage infection remains poorly understood. Here, we analysed the infection of Serratia by a siphovirus (JS26) and the transcriptomic response with, or without type I-E or I-F CRISPR-Cas immunity. In non-immune Serratia, phage infection altered bacterial metabolism by upregulating anaerobic respiration and amino acid biosynthesis genes, while flagella production was suppressed. Furthermore, phage proliferation required a late-expressed viral Cas4 homologue, which did not influence CRISPR adaptation. While type I-E and I-F immunity provided robust defence against phage infection, phage development still impacted the bacterial host. Moreover, DNA repair and SOS response pathways were upregulated during type I immunity. We also discovered that the type I-F system is controlled by a positive autoregulatory feedback loop that is activated upon phage targeting during type I-F immunity, leading to a controlled anti-phage response. Overall, our results provide new insight into phage-host dynamics and the impact of CRISPR immunity within the infected cell.

CRISPR–Cas systems provide bacteria with adaptive immunity against phages and plasmids; however, pathways regulating their activity are not well defined. We recently developed a high-throughput genome-wide method (SorTn-seq) and used this to uncover CRISPR–Cas regulators. Here, we demonstrate that the widespread Rsm/Csr pathway regulates the expression of multiple CRISPR–Cas systems in Serratia (type I-E, I-F and III-A). The main pathway component, RsmA (CsrA), is an RNA-binding post-transcriptional regulator of carbon utilisation, virulence and motility. RsmA binds cas mRNAs and suppresses type I and III CRISPR–Cas interference in addition to adaptation by type I systems. Coregulation of CRISPR–Cas and flagella by the Rsm pathway allows modulation of adaptive immunity when changes in receptor availability would alter susceptibility to flagella-tropic phages. Furthermore, we show that Rsm controls CRISPR–Cas in other genera, suggesting conservation of this regulatory strategy. Finally, we identify genes encoding RsmA homologues in phages, which have the potential to manipulate the physiology of host bacteria and might provide an anti-CRISPR activity.

Brevibacillus laterosporus (Bl) is a Gram-positive and spore-forming bacterium belonging to the Brevibacillus brevis phylogenetic cluster. Globally, insect pathogenic strains of the bacterium have been isolated, characterised, and some activities patented. Two isolates, Bl 1821L and Bl 1951, exhibiting pathogenicity against the diamondback moth and mosquitoes, are under development as a biopesticide in New Zealand. However, due to the suspected activity of putative antibacterial proteins (ABPs), the endemic isolates often grow erratically. Various purification methods including size exclusion chromatography, sucrose density gradient centrifugation, polyethylene glycol precipitation, and ammonium sulphate precipitation employed in this study enabled the isolation of two putative antibacterial proteins of ~30 kD and ~48 kD from Bl 1821L and one putative antibacterial protein of ~30 kD from Bl 1951. Purification of the uninduced cultures of Bl 1821L and Bl 1951 also yielded the protein bands of ~30 kD and ~48 kD on SDS-PAGE which indicated their spontaneous induction. Disc diffusion assay was used to determine the antagonistic activities of the putative ABPs. Subsequent transmission electron microscope (TEM) examination of purified putative antibacterial protein-containing solution showed the presence of encapsulin (~30 kD) and polysheath (~48 kD) like structures. Although only the ~30 kD protein was purified from Bl 1951, both structures were seen in this strain under TEM. Furthermore, while assessing the antibacterial activity of some fractions of Bl 1951 against Bl 1821L in size exclusion chromatography method, population of Bl 1821L persister cells was noted. Overall, this work added a wealth of knowledge for the purification of the HMW proteins (bacteriocins) of the Gram-positive bacteria including Bl.

Bacteria have diverse defenses against phages. In response, jumbo phages evade multiple DNA-targeting defenses by protecting their DNA inside a nucleus-like structure. We previously demonstrated that RNA-targeting type III CRISPR-Cas systems provide jumbo phage immunity by recognizing viral mRNA exported from the nucleus for translation. Here, we demonstrate that recognition of phage mRNA by the type III system activates a cyclic triadenylate-dependent accessory nuclease, NucC. Although unable to access phage DNA in the nucleus, NucC degrades the bacterial chromosome, triggers cell death, and disrupts phage replication and maturation. Hence, type-III-mediated jumbo phage immunity occurs via abortive infection, with suppression of the viral epidemic protecting the population. We further show that type III systems targeting jumbo phages have diverse accessory nucleases, including RNases that provide immunity. Our study demonstrates how type III CRISPR-Cas systems overcome the inaccessibility of jumbo phage DNA to provide robust immunity.

Take-all, caused by the fungal pathogen Gaeumannomyces graminis var. tritici (Ggt) is an important root disease of cereals and grasses. An increasing occurrence of very light seeds in some New Zeal...

The annual regeneration from seeds of subterranean clover plants after initial sowing depends on the hardseededness (HSmax, %), its breakdown rate (HSbreak, %/ °C) and the germinability of the seed...

Toxin-antitoxin systems play key roles in bacterial adaptation, including protection from antibiotic assault and infection by bacteriophages. The type IV toxin-antitoxin system AbiE encodes a DUF1814 nucleotidyltransferase-like toxin, and a two-domain antitoxin. In Streptococcus agalactiae, the antitoxin AbiEi negatively autoregulates abiE expression through positively co-operative binding to inverted repeats within the promoter. The human pathogen Mycobacterium tuberculosis encodes four DUF1814 putative toxins, two of which have antitoxins homologous to AbiEi. One such M. tuberculosis antitoxin, named Rv2827c, is required for growth and whilst the structure has previously been solved, the mode of regulation is unknown. To complete the gaps in our understanding, we first solved the structure of S. agalactiae AbiEi to 1.83 Å resolution for comparison with M. tuberculosis Rv2827c. AbiEi contains an N-terminal DNA binding domain and C-terminal antitoxicity domain, with bilateral faces of opposing charge. The overall AbiEi fold is similar to Rv2827c, though smaller, and with a 65° difference in C-terminal domain orientation. We further demonstrate that, like AbiEi, Rv2827c can autoregulate toxin-antitoxin operon expression. In contrast with AbiEi, the Prv2827c promoter contains two sets of inverted repeats, which bind Rv2827c with differing affinities depending on the sequence consensus. Surprisingly, Rv2827c bound with negative co-operativity to the full Prv2827c promoter, demonstrating an unexpectedly complex form of transcriptional regulation.

Bacteria are under immense evolutionary pressure from their viral invaders-bacteriophages. Bacteria have evolved numerous immune mechanisms, both innate and adaptive, to cope with this pressure. The discovery and exploitation of CRISPR-Cas systems have stimulated a resurgence in the identification and characterization of anti-phage mechanisms. Bacteriophages use an extensive battery of counter-defence strategies to co-exist in the presence of these diverse phage defence mechanisms. Understanding the dynamics of the interactions between these microorganisms has implications for phage-based therapies, microbial ecology and evolution, and the development of new biotechnological tools. Here we review the spectrum of anti-phage systems and highlight their evasion by bacteriophages.

When successful, the operation of local and international networks of crop seed distribution or “seed systems” ensures farmer access to seed and impacts rural livelihoods and food security. Farmers are both consumers and producers in seed systems and benefit from access to global markets. However, phytosanitary measures and seed purity tests are also needed to maintain seed quality and prevent the spread of costly weeds, pests and diseases, in some countries regulatory controls have been in place since the 1800s. Nevertheless, seed contaminants are internationally implicated in between 7% and 37% of the invasive plant species and many of the agricultural pests and diseases. We assess biosecurity risk across international seed trade networks of forage crops using models of contaminant spread that integrate network connectivity and trade volume. To stochastically model hypothetical contaminants through global seed trade networks, realistic dispersal probabilities were estimated from quarantine weed seed detections and incursions from border security interception data in New Zealand. For our test case we use contaminants linked to the global trade of ryegrass and clover seed. Between 2014 and 2018 only four quarantine weed species (222 species and several genera are on the quarantine schedule) warranting risk mitigation were detected at the border. Quarantine weeds were rare considering that average import volumes were over 190 tonnes for ryegrass and clover, but 105 unregulated contaminant species were allowed in. Ryegrass and clover seed imports each led to one post-border weed incursion response over 20 years. Trade reports revealed complex global seed trade networks spanning >134 (ryegrass) and >110 (clover) countries. Simulations showed contaminants could disperse to as many as 50 (clover) or 80 (ryegrass) countries within 10 time-steps. Risk assessed via network models differed 18% (ryegrass) or 48% (clover) of the time compared to risk assessed on trade volumes. We conclude that biosecurity risk is driven by network position, the number of trading connections and trade volume. Risk mitigation measures could involve the use of more comprehensive lists of regulated species, comprehensive inspection protocols, or the addition of field surveillance at farms where seed is planted.

Perennial ryegrass (Lolium perenne) is the major pasture grass in New Zealand but is highly susceptible to take-all disease, caused by the root-rot pathogen Gaeumannomyces graminis (Gg). Isolates of the fungus Trichoderma atroviride are known to control Gg but it is not known if a mixture of isolates would be more effective than individual ones. Soil from a field naturally infested with Gg was placed in containers in a glasshouse and sown with ryegrass seeds then treated with one of three Trichoderma atroviride isolates or a mixture of all three isolates. All T. atroviride treatments significantly increased shoot dry matter by 46–73% and root dry matter by 42–62% compared with the control but a mixture of isolates was no more effective than individual isolates. Application of T. atroviride also significantly decreased root disease severity, which was negatively correlated with root dry matter. Takeall in pastures could possibly be controlled by overdrilling grass with a single isolate of T. atroviride.

Plutella xylostella (diamondback moth) is a prominent pest of brassicas which is now resistant to most insecticides. Despite years of research, the range of available products used in biological control of diamondback moth is still somewhat limited. We isolated putative endophytic fungi from New Zealand cabbage plants to search for unique biological control agents of diamondback moth larvae. The larvae were fed leaf discs from commercially grown cabbage covered in spores from endophytic fungal isolates to test the insecticidal properties of these fungi. Twenty of the 52 fungal isolates tested failed to kill any diamondback moth larvae. However, three isolates of Lecanicillium muscarium induced mortality greater than 80%. While these isolates have potential for use in biological control applications, further research into propagation, formulation, and method, rate and timing of application is needed.

Some entomopathogenic fungi such as Metarhizium and Beauveria not only have the ability to infect and kill insects but also the capability to associate with plant roots. The potential benefits from...

Changes in cardiac function that occur with exercise training have been studied in detail, but those accompanying evolved increases in the duration or intensity of physical activity are poorly understood. To address this gap, we studied electrocardiograms (ECGs) of mice from an artificial selection experiment in which four replicate lines are bred for high voluntary wheel running (HR) while four non-selected lines are maintained as controls (C). ECGs were recorded using an ECGenie (Mouse Specifics, Inc.) both before and after six days of wheel access (as used in the standard protocol to select breeders). We hypothesized that HR mice would show innate differences in ECG characteristics and that the response to training would be greater in HR mice relative to C mice because the former run more. After wheel access, in statistical analyses controlling for variation in body mass, all mice had lower heart rates, and mice from HR lines had longer PR intervals than C lines. Also after wheel access, male mice had increased heart rate variability, whereas females had decreased heart rate variability. With body mass as a covariate, six days of wheel access significantly increased ventricle mass in both HR and C males. Within the HR lines, a subset of mice known as mini-muscle individuals have a 50% reduction in hindlimb muscle mass and generally larger internal organs, including the heart ventricles. As compared with normal-muscled individuals, mini-muscle individuals had a longer QRS complex, both before and after wheel access. Some studies in other species of mammals have shown correlations between athletic performance and QRS duration. Correlations between wheel running and either heart rate or QRS duration (before wheel running) among the eight individual lines of the HR selection experiment or among 17 inbred mouse strains taken from the literature were not statistically significant. However, total revolutions and average speed were negatively correlated with PR duration among lines of the HR selection experiment for males, and duration of running was negatively correlated with PR duration among 17 inbred strains for females. We conclude that HR mice have enhanced trainability of cardiac function as compared with C mice (as indicated by their longer PR duration after wheel access), and that the mini-muscle phenotype causes cardiac changes that have been associated with increased athletic performance in previous studies of mammals.

The ability of disease-causing bacteria to synergise infection by the entomopathogen Metarhizium of larvae of Costelytra giveni, a New Zealand native scarab, has been previously demonstrated, but the role of soil bacteria in infections is unknown. Bacteria isolated from both dead C. giveni larvae and soil were assessed for their effect on the virulence of Metarhizium novozealandicum C14 towards C. giveni larvae. Seven bacterial isolates showed synergism with M. novozealandicum C14 against second instar larvae, but no synergism was found against third instar larvae. No apparent disease was noted with C. giveni larvae independently challenged with the bacteria isolates. An isolate of Yersinia enterocolitica (isolate 6-1) in combination with M. novozealandicum C14 caused higher larval mortality than the other bacterial isolate-entomopathogen combinations against second instar larvae. Using plate assays, Y. enterocolitica 6-1 was found to produce more chitinase, but not protease, compared with the other bacterial strains suggesting a role of chitinase in synergism. This study suggests that the combination of Y. enterocolitica (isolate 6-1) and M. novozealandicum C14 could provide effective biocontrol of second instar grass grub larvae.

Imports of seeds for sowing are a major pathway for the introduction of contaminant seeds, and many agricultural weeds globally naturalised originally have entered through this pathway. Effective management of this pathway is a significant means of reducing future plant introductions and helps minimise agricultural losses. Using a national border inspection database, we examined the frequency, origin and identity of contaminant seeds within seed for sowing shipments entering New Zealand between 2014-2018. Our analysis looked at 41,610 seed lots across 1,420 crop seed species from over 90 countries. Overall, contamination was rare, occurring in 1.9% of all seed lots. Among the different crop types, the arable category had the lowest percentage of seed lots contaminated (0.5%) and the forage category had the highest (12.6%). Crop seeds Capsicum , Phaseolus and Solanum had the lowest contamination rates (0.0%). Forage crops Medicago (27.3%) and Trifolium (19.8%) had the highest contamination rates. Out of 191 genera recorded as contaminants, Chenopodium was the most common. Regulated quarantine weeds were the rarest contaminant type, only occurring in 0.06% of seed lots. Sorghum halepense was the most common quarantine species and was only found in vegetable seed lots. Vegetable crop seed lots accounted for approximately half of all quarantine species detections, Raphanus sativus being the most contaminated vegetable crop. Larger seed lots were significantly more contaminated and more likely to contain a quarantine species than smaller seed lots. These findings support International Seed Testing Association rules on maximum seed lot weights. Low contamination rates suggest industry practices are effective in minimising contaminant seeds. Considering New Zealand inspects every imported seed 3 lot, utilises a working sample size 5 times larger than International Seed Testing Association rules require, trades crop seed with approximately half of the world's countries and imports thousands of crop seed species, our study provides a unique overview of contaminant seeds that move throughout the seed for sowing system.

Bacteriophage defences are divided into innate and adaptive systems. Serratia sp. ATCC 39006 has three CRISPR-Cas adaptive immune systems, but its innate immune repertoire is unknown. Here, we re-sequenced and annotated the Serratia genome and predicted its toxin–antitoxin (TA) systems. TA systems can provide innate phage defence through abortive infection by causing infected cells to ‘shut down’, limiting phage propagation. To assess TA system function on a genome-wide scale, we utilized transposon insertion and RNA sequencing. Of the 32 TA systems predicted bioinformatically, 4 resembled pseudogenes and 11 were demonstrated to be functional based on transposon mutagenesis. Three functional systems belonged to the poorly characterized but widespread, AbiE, abortive infection/TA family. AbiE is a type IV TA system with a predicted nucleotidyltransferase toxin. To investigate the mode of action of this toxin, we measured the transcriptional response to AbiEii expression. We observed dysregulated levels of tRNAs and propose that the toxin targets tRNAs resulting in bacteriostasis. A recent report on a related toxin shows this occurs through addition of nucleotides to tRNA(s). This study has demonstrated the utility of functional genomics for probing TA function in a high-throughput manner, defined the TA repertoire in Serratia and shown the consequences of AbiE induction.

The components of seed quality essentially fall into three categories: accurate description, hygiene, and viability and potential performance (Coolbear and Hill, 1988). Any complete assessment of the third category should consider the capacity of the seed to produce normal seedlings expected field emergence and uniformity potential storability To obtain this information seed is tested for viability, and increasingly, for vigor. The concepts and underlying mechanisms of seed viability and seed vigor have been presented in Chapters 1 and 2. This chapter will deal with the measurement of seed viability and vigor and examine the methods available for determining seedlot potential and actual performance in the field and during storage.

Hepatocellular carcinoma (HCC) develops in the context of chronic inflammatory liver disease and has an extremely poor prognosis. An immunosuppressive tumor microenvironment may contribute to therapeutic failure in metastatic HCC. Here, we identified unique molecular signatures pertaining to HCC disease progression and tumor immunity by analyzing genome-wide RNA-Seq data derived from HCC patient tumors and non-tumor cirrhotic tissues. Unsupervised clustering of gene expression data revealed a gradual suppression of local tumor immunity that coincided with disease progression, indicating an increasingly immunosuppressive tumor environment during HCC disease advancement. IHC examination of the spatial distribution of CD8+ T cells in tumors revealed distinct intra- and peri-tumoral subsets. Differential gene expression analysis revealed an 85-gene signature that was significantly upregulated in the peri-tumoral CD8+ T cell-excluded tumors. Notably, this signature was highly enriched with components of underlying extracellular matrix, fibrosis, and epithelial–mesenchymal transition (EMT). Further analysis condensed this signature to a core set of 23 genes that are associated with CD8+ T cell localization, and were prospectively validated in an independent cohort of HCC specimens. These findings suggest a potential association between elevated fibrosis, possibly modulated by TGF-β, PDGFR, SHH or Notch pathway, and the T cell-excluded immune phenotype. Indeed, targeting fibrosis using a TGF-β neutralizing antibody in the STAM™ model of murine HCC, we found that ameliorating the fibrotic environment could facilitate redistribution of CD8+ lymphocytes into tumors. Our results provide a strong rationale for utilizing immunotherapies in HCC earlier during treatment, potentially in combination with anti-fibrotic therapies., Liver cancer: Poor tumor immunity linked to fibrosis Anti-fibrotic drugs could help make patients with liver cancer more responsive to immune-targeted therapies. A team led by Shih-Min Huang and Mark Lackner from Genentech in South San Francisco, CA, USA, analyzed gene expression profiles in tumor samples taken from 98 people with hepatocellular carcinoma, the most common form of liver cancer. The expression analysis revealed a persistently down-modulated immune microenvironment—and the more advanced the disease, the stronger the immune suppression. The researchers also saw an association between increased expression of genes involved in fibrotic tissue scarring and diminished immune function. They therefore administered an antibody drug that blocks a key growth factor implicated in fibrosis to a mouse model of the disease. These animals showed enhanced immune function, raising the possibility of using a similar strategy in patients undergoing immunotherapy for liver cancer.

Climate change is predicted to increase the frequency of heat stress events during seed production. In two consecutive seasons, plants within a forage rape (Brassica napus L.) seed crop were covered with plastic sheeting to increase ambient air temperature for the period between seed filling (80% seed moisture content) and seed physiological maturity (50% seed moisture content) = T1, between physiological maturity and harvest (14% seed moisture content) = T2, and between 80% seed moisture content and harvest = T3. This resulted in 47 and 102 h when air temperature exceeded 25 °C for T1, 121 and 173 h for T2, and 145 and 228 h for T3 compared with 9 and 30 h for the uncovered control in each season respectively. Hourly thermal time (T base = 25 °C) was calculated for each treatment. Both T1 and T2 resulted in small (2–10%) reductions in germination in each season, but when season data were meaned and analysed, only T2 and T3 significantly reduced germination. Seed mass (as measured by thousand seed weight) was significantly reduced by T1, but not T2, while seed vigour, as assessed by the accelerated ageing and conductivity tests, was significantly reduced by all three treatments, with T3 > T2 > T1. The number of hours that temperature exceeded 25 °C was negatively correlated with germination and seed vigour, but not seed mass. Approximately 100 h of temperature exceeding 25 °C, or an hourly thermal time of 300 °C h (Tb = 25 °C) were required to reduce the vigour status of the seed lot. Crop management strategies to avoid heat stress during seed development are unlikely to succeed in this environment.

Urea, the most commonly used nitrogen fertiliser in New Zealand, can be quickly lost from the system via ammonia volatilisation or nitrate leaching following hydrolysis of urea by urease producing soil microorganisms (UPSMs). This study investigated UPSMs involved in urea degradation for upcoming research to reduce soil urease activity. Soils from under dairy pasture management across New Zealand, with a pasture species component of ryegrass (Lolium perenne L.) and white clover (Trifolium repens L.) and aged between 9 months to 60 years old, were collected, and UPSMs were isolated and identified using both polymerase chain reaction (PCR)-based molecular and conventional methods. The fungal genera belonged to diverse taxonomical groups including the phylum Ascomycota: class: Dothideomycetes, Eurotiomycetes, Leotiomycetes and Sordariomycetes, the phylum Basidiomycota: class: Tremellomycetes and the phylum Zygomycota: order: Mucorales, all of which have a role in urea degradation in soil. Pasture soil-resident urease producing bacteria belonged to the Gammaproteobacteria and Betaproteobacteria. Cupriavidus sp. and Mucor hiemalis showed strong urease activity when cultured on urease medium. This is the first report on the urease activity of the pasture soil inhabitants Pochonia bulbillosa, Mariannaea elegans and Gliomastix sp. This study was part of a larger study underway to investigate control of UPSMs in soil to improve the efficiency of urea utilisation.

In two consecutive seasons, forage rape (Brassica napus L.) plants were exposed to short periods (240°C hr) of heat stress (30°C day/25°C night) during seed filling (80% seed moisture content = S1), at physiological maturity (50% seed moisture content = S2) and at both S1 plus S2 (=S3) in a Biotron before being returned to the field until seed harvest. Seeds were hand harvested at 14% seed moisture content and their quality assessed by measuring germination, seed vigour (using the accelerated ageing and conductivity tests) and seed mass (as determined by thousand seed weight). Heat stress at both S1 and S2 caused a small ( S2>S1. Seed mass was reduced by heat stress at S1 but not at S2. Variable seed vigour in high‐germinating New Zealand‐produced forage rapeseed lots is most likely explained by short periods of heat stress during seed development.

The efficacy of a direct-fired, tractor-mounted steam weeder was compared with that of a flame weeder. At a tractor speed of 3 km h−1, steam killed 100% of 10-day-old mustard plants, sown as a surrogate weed, and flame killed 92%. Plant survival increased with increasing tractor speed, but was always lower for the steam weeder. Mean survival over all speeds was 40% for steam and 74% for flame. By contrast, for 15 agricultural weed species, at a tractor speed of 3 km h−1 weed survival for both weeders was similar. At 3 km h−1 both methods killed Stellaria media and Veronica agrestis at all growth stages assessed, but neither killed Lolium perenne. Plants of all the other species were killed when the treatments were applied soon after emergence (cotyledon stage), but as the plants increased in size they became more resistant to heat, either because the terminal or axillary meristems were protected by soil, or because meristems were protected by thickened stems and leaves as the plants aged.

Many cool-season grasses form permanent, mutualistic symbioses with asexual Epichloë endophytes. These fungal symbionts often perform a protective role within the association as many strains produce secondary metabolites that deter certain mammalian and invertebrate herbivores. Although initially a serious issue for agriculture, due to mammalian toxins that manifested in major animal health issues, selected strains that provide abiotic stress protection to plants with minimal ill effects to livestock are now commercialized and routinely used to enhance pasture performance in many farming systems. These fungal endophytes and their grass hosts have coevolved over millions of years, and it is now generally accepted that most taxonomic groupings of Epichloë are confined to forming compatible associations (i.e., symptomless associations) with related grass genera within a tribe. The most desired compounds associated with Epichloë festucae var. lolii, an endophyte species associated with perennial ryegrass, are peramine and epoxy-janthitrems. No other major secondary metabolites with invertebrate bioactivity have been identified within this association. However, other agriculturally beneficial compounds, such as lolines, have been discovered in related endophyte species that form associations with fescue grasses. A rationale therefore existed to develop novel grass-endophyte associations between loline-producing endophytes originally isolated from tall fescue with elite cultivars of perennial ryegrass to achieve a wider spectrum of insect bioactivity. A suitable loline-producing endophyte strain of Epichloë sp. FaTG-3 was selected and inoculated into perennial ryegrass. We hypothesed that endophyte transmission frequency, endophyte mycelial biomass and endophyte-derived alkaloid production would differ between the original tall fescue host and the artificial association. Consistent with our hypothesis, our data strongly suggest that plant species significantly affected the plant-endophyte association. This effect became more apparent for transmission frequency and endophyte biomass as the plants matured. Overall, the viable endophyte infection frequency was greater in the tall fescue host than in perennial ryegrass, at all sampling dates. Additionally, temperature was found to be a significant factor affecting endophyte transmission frequency, endophyte mycelial biomass and alkaloid production. Implications for the development of novel grass-endophyte associations are discussed.

Bacteria are under immense evolutionary pressure from their viral invaders-bacteriophages. Bacteria have evolved numerous immune mechanisms, both innate and adaptive, to cope with this pressure. The discovery and exploitation of CRISPR-Cas systems have stimulated a resurgence in the identification and characterization of anti-phage mechanisms. Bacteriophages use an extensive battery of counter-defence strategies to co-exist in the presence of these diverse phage defence mechanisms. Understanding the dynamics of the interactions between these microorganisms has implications for phage-based therapies, microbial ecology and evolution, and the development of new biotechnological tools. Here we review the spectrum of anti-phage systems and highlight their evasion by bacteriophages.

CRISPR-Cas adaptive immune systems protect bacteria from phage predation, and other foreign genetic elements such as plasmids. Significant advances have been made regarding how CRISPR-Cas systems elicit immunity; however, comparatively little is known about their regulation. To study CRISPR-Cas regulation, we describe the construction of suicide lacZ-reporter plasmids with different antibiotic resistance cassettes. Through recombination into the host chromosome, single-copy expression can be achieved, thus preserving natural gene expression and maintaining a reporter expression output that reflects regulation within a normal genomic context. Previous work determined that the galactose metabolism gene galM, decreased the expression of the cas operon in Pectobacterium atrosepticum. We used the new integrative reporters to investigate galK, a gene that is located elsewhere in the genome and is responsible for the conversion of α-D-Galactose to Galactose-1-P during galactose metabolism. Deletion of galK led to elevated cas expression in a CRP-dependent manner but had no effect on CRISPR array expression. These results highlight that the metabolic status of the host cell is linked to the induction of CRISPR-Cas immunity.