Construction of an Infectious DNA Clone of Grapevine Geminivirus A Isolate GN and Its Biological Activity in Plants Analyzed Using an Efficient and Simple Inoculation Method.

The pathogenicity of grapevine geminivirus A (GGVA), a recently identified DNA virus, to grapevine plants remains largely unclear. Here, we report a new GGVA isolate (named GGVAQN) obtained from grapevine 'Queen Nina' plants with severe disease symptoms. The infectious clone of GGVAQN (pXT-GGVAQN) was constructed to investigate its pathogenicity. Nicotiana benthamiana plants inoculated with GGVAQN by agroinfiltration displayed upward leaf curling and chlorotic mottling symptoms. A simple, quick, and efficient method for delivering DNA clones of GGVAQN into grapevine plants was developed, by which Agrobacterium tumefaciens cells carrying pXT-GGVAQN were introduced into the roots of in vitro-grown 'Red Globe' grape plantlets with a syringe. By this method, all 'Red Globe' grape plants were systemically infected with GGVAQN, and the plants exhibited chlorotic mottling symptoms on their upper leaves and downward curling, interveinal yellowing, and leaf-margin necrosis symptoms on their lower leaves. Our results provide insights into the pathogenicity of GGVA and a simple and efficient inoculation method to deliver infectious viral clones to woody perennial plants.

Identification of a suitable method of inoculation for reducing background effect in mock-inoculated controls during gene expression studies in Arabidopsis thaliana.

The recent advancement in the field of transcriptome and methylome sequencing helped scientists to analyse the gene expression and epigenetic status of different genes. Several genes and their regulatory pathways have been discovered due to research into plant-microbe interactions. Previous research on plant-Agrobacterium interactions found that the method of inoculation (wounding using a syringe), resulted in altered DNA methylation of the host DNA repair gene promoters. The expression study of host defence genes revealed that the method of inoculation masked the host response to bacteria. It could be possible that these method-induced changes could interfere with various defence regulatory pathways, which otherwise would not be triggered by the bacteria alone. Hence, it would be critical to identify an appropriate method of inoculation that could provide more unambiguous interpretation of studies involving gene expression and regulation in plants under bacterial stress. The expression dynamics of two defence genes, PR1 and NPR1, under various combinations of parameters such as three different methods of inoculation, treatment with five different bacterial re-suspending solutions, and at three different post-inoculation time intervals were examined in the model plant Arabidopsis thaliana. The H2O2 and superoxide (O2-) production due to various inoculation methods and re-suspending solutions on the host was also studied. The flood inoculation method, which used sterile deionized water (SDW) to re-suspend bacteria, elicited the slightest response in mock-inoculated plants. Under this method, Agrobacterium strains carrying the GUS reporter gene were used to test bacterial infectivity. Blue sectors were found in plants infected for 24 and 48 h. PR1 and NPR1 expression were significantly altered at various time intervals after inoculation. So, for experiments involving Arabidopsis-Agrobacterium interaction with minimal background influences, such as gene expression and epigenetic analyses, the flood inoculation method using SDW as the resuspension liquid is proposed. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01381-x.

An Effective Method of Ribes spp. Inoculation with Blackcurrant Reversion Virus under In Vitro Conditions.

Blackcurrant reversion virus (BRV) is the most destructive currant-infecting and mite-transmitted pathogen from the genus Nepovirus. In this work, BRV transmission in the system Ribes ex vitro-Ribes in vitro was applied for the first time. Triple infection of BRV identified in blackcurrant cv. Gojai was used for phylogenetic analysis and inoculation assay. Transmission of BRV was successful due to its stability in the inoculum for up to 8 days at 4 °C; all BRV isolates were infectious. Our suggested inoculation method through roots was applied in six Ribes spp. genotypes with 100.0% reliability, and the expression levels of defence-related gene PR1 to biotic stress was observed. The prevalence of the virus in microshoots after 2-14 days post-inoculation (dpi) was established by PCR. In resistant genotypes, the BRV was identified up to 8 dpi; meanwhile, infection remained constant in susceptible genotypes. We established that BRV transmission under controlled conditions depends on the inoculum quality, post-inoculation cultivation temperature, and host-plant susceptibility to pathogen. This in vitro inoculation method opens possibilities to reveal the resistance mechanisms or response pathways to BRV and can be used for the selection of resistant Ribes spp. in breeding programs.

Comparison of Hyphal Fragments and Spores to Evaluate the Pathogenicity of the Eucalyptus Leaf and Shoot Pathogen Calonectria pseudoreteaudii.

The genus Calonectria includes many aggressive plant-pathogenic species with a worldwide distribution. Calonectria leaf blight is one of the most prominent diseases of Eucalyptus trees in Southeast Asian and South American plantations. Inoculation trials to evaluate pathogenicity of Calonectria spp. typically use conidial suspensions but this is not possible for species that do not sporulate sufficiently in culture. Calonectria pseudoreteaudii is one of the species that is most aggressive to Eucalyptus in China but most isolates fail to produce conidia in culture, requiring an alternative procedure for artificial inoculation. This study compared inoculations utilizing conidial and hyphal fragment suspensions. Two Eucalyptus genotypes were used, and these were inoculated with different concentrations of hyphal fragments or conidia of three C. pseudoreteaudii isolates. Three days after inoculation, the treated Eucalyptus plants displayed similar disease symptoms, regardless of whether they had been inoculated with conidia or hyphal fragments. This was consistent for all C. pseudoreteaudii isolates and also the different Eucalyptus genotypes. The results demonstrate that hyphal fragment suspensions can be used to provide a reliable indication of C. pseudoreteaudii isolate pathogenicity when conidia are not available for inoculation studies.

Establishment of an Artificial Inoculation Method of Ustilaginoidea virens Without Damaging the Rice Panicle Sheaths.

Rice false smut (RFS) is a destructive disease of rice worldwide caused by Ustilaginoidea virens. Nevertheless, there is a lack of efficient and stable artificial inoculation method to simulate the natural infection of U. virens, which is an important factor limiting further research on the pathogen. The purpose of this study was to establish an artificial inoculation method, which can simulate the natural infection process of U. virens without destroying the panicle sheath structure of rice. In this research, rice plants were inoculated by soaking roots at the seedling stage, spraying at the tillering stage, injecting at the booting stage, and again spraying at the flowering stage to determine the appropriate artificial inoculation time. Meanwhile, the panicle sheath instillation method and the injection inoculation method were compared. The results show that stages 6 to 8 of young panicle differentiation are an important period for U. virens infection. There were no significant differences in the mean rates of infected panicles, mean rates of infected grains, and maximum infected grains per panicle between the two inoculation methods. However, the frequency of RFS ball occurrence at the upper part of the panicles was significantly higher on the spikelets inoculated by the injection method than that of spikelets inoculated by natural infection and panicle sheath instillation. Therefore, panicle sheath instillation method was more similar to the natural infection of U. virens in the field. This research exhibited an innovative artificial inoculation method for identification of U. virens pathogenicity and evaluation of rice resistance against RFS.

Wet versus Dry Inoculation Methods Have a Significant Effect of Listeria monocytogenes Growth on Many Types of Whole Intact Fresh Produce.

ABSTRACT: Listeria monocytogenes causes relatively few outbreaks linked to whole fresh produce but triggers recalls each year in the United States. There are limited data on the influence of wet versus dry inoculation methods on pathogen growth on whole produce. A cocktail of five L. monocytogenes strains that included clinical, food, and environmental isolates associated with foodborne outbreaks and recalls was used. Cultures were combined to target a final wet inoculum concentration of 4 to 5 log CFU/mL. The dry inoculum was prepared by mixing wet inoculum with 100 g of sterile sand and drying for 24 h. Produce investigated belonged to major commodity families: Ericaceae (blackberry, raspberry, and blueberry), Rutaceae (lemon and mandarin orange), Rosaceae (sweet cherry), Solanaceae (tomato), Brassaceae (cauliflower and broccoli), and Apiaceae (carrot). Whole intact, inoculated fruit and vegetable commodities were incubated at 2, 12, 22, and 35 ± 2°C. Commodities were sampled for up to 28 days, and the experiment was replicated six times. The average maximum growth increase was obtained by measuring the maximum absolute increase for each replicate within a specific commodity, temperature, and inoculation method. Data for each commodity, replicate, and temperature were used to create primary growth or survival models describing the lag phase and growth or shoulder and decline as a function of time. Use of a liquid inoculum (versus dry inoculum) resulted in a markedly increased L. monocytogenes growth rate and growth magnitude on whole produce surfaces. Temperature highly influenced this difference: a greater effect seen with more commodities at higher temperatures (22 and 35°C) versus lower temperatures (2 and 12°C). These findings need to be explored for other commodities and pathogens. The degree to which wet or dry inoculation techniques more realistically mimic contamination conditions throughout the supply chain (e.g., production, harvest, postharvest, transportation, or retail) should be investigated.

Models for factors influencing pathogen survival in low water activity foods from literature data are highly significant but show large unexplained variance.

Factors that control pathogen survival in low water activity foods are not well understood and vary greatly from food to food. A literature search was performed to locate data on the survival of foodborne pathogens in low-water activity (<0.70) foods held at temperatures <37 °C. Data were extracted from 67 publications and simple linear regression models were fit to each data set to estimate log linear rates of change. Multiple linear stepwise regression models for factors influencing survival rate were developed. Subset regression modeling gave relatively low adjusted R2 values of 0.33, 0.37, and 0.48 for Salmonella, E. coli and L. monocytogenes respectively, but all subset models were highly significant (p < 1.0e-9). Subset regression models showed that Salmonella survival was significantly (p < 0.05) influenced by temperature, serovar and strain type, water activity, inoculum preparation method, and inoculation method. E. coli survival was significantly influenced by temperature, water activity, and inoculum preparation. L. monocytogenes survival was significantly influenced by temperature, serovar and strain type, and inoculum preparation method. While many factors were highly significant (p < 0.001), the high degrees of variability show that there is still much to learn about the factors which govern pathogen survival in low water activity foods.

Screening fungal endophytes from a wild grass for growth promotion in tritordeum, an agricultural cereal.

Celtica gigantea(= Stipa gigantea) is a large perennial grass which grows in nutrient-poor sandy soils in semiarid zones of the western Iberian Peninsula. The purpose of this work was to find out if culturable fungal symbionts isolated from roots of this wild grass could have growth promoting activity in tritordeum, a hybrid cereal for human consumption. A survey of fungi from the root endosphere of C. gigantea produced an isolate collection consisting of 60 different taxa, mostly ascomycetes. Fungal strains were inoculated into tritordeum plants in order to evaluate their effect in leaf and root biomass, nutrient content, and total antioxidant capacity. Two consecutive screening processes were made to test endophyte effects in plants. In the first screening, 66 strains were inoculated into seedlings by dipping roots in a liquid suspension of inoculum. In the second screening, 13 strains selected from the first screening were inoculated by sowing seeds in a substrate containing inoculum. The inoculation method used in the second screening involved less labor and plant manipulation and improved the quantity and quality of the inoculum, making it more appropriate for big scale experimental inoculation procedures. Several fungal strains promoted leaf or root growth. In particular, a strain belonging to the genus Diaporthe caused an increase in leaf and root biomass in both screening processes, suggesting that this endophyte might have a good potential for field application in tritordeum.

A Greenhouse Method to Evaluate Sunflower Quantitative Resistance to Basal Stalk Rot Caused by Sclerotinia sclerotiorum.

Resistance of sunflower to basal stalk rot (BSR) caused by the fungus Sclerotinia sclerotiorum is quantitative, controlled by multiple genes contributing small effects. Consequently, artificial inoculation procedures allowing sufficient throughput and resolution of resistance are needed to identify highly resistant sunflower germplasm resources and to map loci contributing to resistance. The objective of this study was to develop a greenhouse-based method for evaluating sunflower quantitative resistance to BSR that would be simple, space- and time-efficient, high throughput, high resolution, and correlated with field observations. Experiments were conducted with 5-week-old sunflower plants and Sclerotinia-infested millet seed as inoculum to assess the impact of pot size and temperature and to determine the most favorable inoculum rate and placement. Subsequently, an additional experiment was performed to assess the correlation of the greenhouse inoculation procedure with field results by using a panel of 32 sunflower genotypes with known field response to BSR previously determined in multiyear, multilocation artificially inoculated trials. Experimental observations indicated that the newly developed greenhouse inoculation procedure provided improved resolution to identify highly resistant genotypes and was strongly correlated with field observations. This method will be useful for screening of sunflower experimental and breeding materials, disease phenotyping of genetic mapping populations, and evaluation of resistance to different pathogen isolates.

Impact of Inoculum Type on the Microbial Community and Power Performance of Urine-Fed Microbial Fuel Cells.

Bacteria are the driving force of the microbial fuel cell (MFC) technology, which benefits from their natural ability to degrade organic matter and generate electricity. The development of an efficient anodic biofilm has a significant impact on the power performance of this technology so it is essential to understand the effects of the inoculum nature on the anodic bacterial diversity and establish its relationship with the power performance of the system. Thus, this work aims at analysing the impact of 3 different types of inoculum: (i) stored urine, (ii) sludge and (iii) effluent from a working MFC, on the microbial community of the anodic biofilm and therefore on the power performance of urine-fed ceramic MFCs. The results showed that MFCs inoculated with sludge outperformed the rest and reached a maximum power output of 40.38 mW·m-2anode (1.21 mW). The power performance of these systems increased over time whereas the power output by MFCs inoculated either with stored urine or effluent decreased after day 30. These results are directly related to the establishment and adaptation of the microbial community on the anode during the assay. Results showed the direct relationship between the bacterial community composition, originating from the different inocula, and power generation within the MFCs.

Improved flavor profiles of red pitaya (Hylocereus lemairei) wine by controlling the inoculations of Saccharomyces bayanus and Metschnikowia agaves and the fermentation temperature.

The effects of the inoculation method of Saccharomyces bayanus BV818 and non-Saccharomyces yeast Metschnikowia agaves P3-3 and the fermentation temperature on the volatile profiles of red pitaya wine were investigated in the present study. Although the growth of P3-3 was inhibited by BV818 in the mixed inoculations, simultaneous and sequential inoculations promoted the production of seven volatiles, including higher alcohols (propan-1-ol, 3-methyl-1-butanol and phenethyl alcohol), esters (ethyl decanoate and diethyl succinate), acid (2-ethylhexanoic acid), and ketone (acetoin). Sequential inoculation produced the largest total content of volatile compounds and exhibited the best in the global aroma. The red pitaya wine produced in different inoculations can be separated by its main volatile components. Furthermore, the highest total content was yielded at 25 °C for alcohols and at 21 °C for esters and acids. Within an experimental range of 17 °C to 29 °C, the contents of benzaldehyde and acetoin decreased with the increase in temperature, whereas the change in 4-ethyl-2-methoxyphenol content was the opposite. The similarly high total contents of volatiles and global aroma score were yielded via sequential inoculation at 21 °C and 25 °C. Therefore, the desired red pitaya wine can be effectively produced by modulating the inoculation method and fermentation temperature.

Development of a Protocol and a Diagrammatic Scale for Quantification of Bacterial Leaf Streak Disease on Young Plants of Maize.

Bacterial leaf streak (BLS), caused Xanthomonas vasicola pv. vasculorum (Xvv), has become a major concern for maize production, mainly in the United States and South America. Therefore, this study aimed to establish a protocol for Xvv inoculation in young maize plants under controlled conditions and to develop and validate a diagrammatic scale for evaluation of maize hybrids in regard to BLS resistance. The study was carried out in three steps: the establishment of a protocol for inoculation of Xvv in young maize plants under controlled conditions; the development and validation of a diagrammatic scale for BLS severity evaluation; and the screening for BLS resistance of 45 hybrids using the proposed protocol for bacterial inoculation and the diagrammatic scale developed in this study. Besides reproducing a more natural Xvv infection, the bacterial suspension spraying without injury inoculation method induced higher disease incidence and severity, as well as reproducibility of results under the experimental conditions established in this study. The proposed diagrammatic scale allowed evaluating BLS severity with up to 97.49% of the leaf area affected by the disease. Further, the use of the diagrammatic scale resulted in an increase of accuracy from 0.909 up to 0.992. The reaction of 45 maize hybrids to BLS allowed establishing six major groups of susceptibility to the disease. The most resistant maize hybrids to BLS formed a group of 13 hybrids, with disease severity below 5%.

A new method for the inoculation of Phytophthora palmivora (Butler) into cacao seedlings under greenhouse conditions.

BACKGROUND: The black pod disease affects cacao plantations worldwide; it is caused by the oomycete species of the genus Phytophthora. The resistance of cacao plants to the black pod is commonly evaluated by artificial inoculation of the pathogen and the monitoring of the disease symptoms. However, it is difficult to identify resistant plants because the commonly used methods for the inoculation of the pathogens produce inconsistent results. Therefore, this study aimed to develop an efficient and reliable method to evaluate the resistance of Theobroma cacao seedlings to the infection by Phytophthora palmivora. RESULTS: Seedlings of different cacao genotypes were inoculated with P. palmivora under greenhouse conditions using the previously reported inoculation methods and a newly proposed method, the agar-water solution method. While none of the previously reported methods was effective, the agar-water solution method ensured a 100% seedling infection under greenhouse conditions. The proposed agar-water methodology is fast, simple and reproducible. Furthermore, the evaluation of this method in susceptible (CCN-51) and tolerant (SCA-6) T. cacao genotypes produced the expected contrasting results. CONCLUSIONS: The agar-water solution method presented in this study is an efficient alternative inoculation protocol for the identification of cacao genotypes that are resistant to black pod under greenhouse conditions.

Research Note: Evaluation of several inoculation procedures for colonization of day-old broiler chicks with Salmonella Heidelberg.

Before starting a study with many birds, it helps to know the method of chick inoculation. The objective was to compare 3 methods of Salmonella challenge (oral gavage [OR], intracloacal inoculation [IC], and seeder bird [SB]). Day-old broiler chicks (n = 100) were inoculated with 106 colony forming units (CFU) per chick of a marker strain of Salmonella Heidelberg (SH) with each route of inoculation. Chicks (n = 25) inoculated by each route were placed in floor pens on fresh pine shavings litter. For the seeder batch, 5 colonized chicks, each orally gavaged with 106 CFUs, were placed with 20 pen mates. Two weeks after inoculation, 10 birds from each pen and the 5 inoculated seeder birds were euthanized, the ceca were aseptically removed and macerated with a rubber mallet and weighed, and 3 times (w/v) buffered peptone was added and stomached for 60 s. Serial dilutions were made and plated onto Brilliant Green Sulfa plates containing 200 ppm nalidixic acid. Plates were incubated along with the stomached ceca for 24 h at 37°C. If no colonies appeared on the plates, an additional plate was streaked from the preenriched bag and incubated for 24 h at 37°C. In addition to all seeder birds being positive, the number of SH-positive birds out of 20 sampled in each group was 13, 17, and 7 for OR, IC, and SB, respectively. The level of SH per g of ceca and cecal contents was log (SE) 3.0 (0.7), 2.0 (0.4), and 2.6 (0.4) for OR, IC, and SB, respectively. After enrichment, the number of colonized birds out of 20 was 18, 20, and 10 for OR, IC, and SB, respectively. In conclusion, this study suggests that IC is the method to use to ensure most of the challenged birds are colonized. However, if you prefer to have a smaller percentage of the birds colonized with higher levels, then OR might be better.

Pathogenesis of Respiratory Syncytial Virus Infection in BALB/c Mice Differs Between Intratracheal and Intranasal Inoculation.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease requiring hospitalization in infants. There are no market-approved vaccines or antiviral agents available, but a growing number of vaccines and therapeutics are in (pre)clinical stages of development. Reliable animal models are crucial to evaluate new vaccine concepts, but in vivo RSV research is hampered by the lack of well-characterized animal models that faithfully mimic the pathogenesis of RSV infection in humans. Mice are frequently used in RSV infection and vaccination studies. However, differences in the use of mouse strains, RSV subtypes, and methodology often lead to divergent study outcomes. To our knowledge, a comparison between different RSV inoculation methods in mice has not been described in the literature, even though multiple methods are being used across different studies. In this study, we evaluated various pathological and immunological parameters in BALB/c mice after intratracheal or intranasal inoculation with RSV-A2. Our study reveals that intranasal inoculation induces robust pathology and inflammation, whereas this is not the case for intratracheal inoculation. As immunopathology is an important characteristic of RSV disease in infants, these data suggest that in mice intranasal inoculation is a more appropriate method to study RSV infection than intratracheal inoculation. These findings will contribute to the rational experimental design of future in vivo RSV experiments.

Standard method for detecting Bombyx mori nucleopolyhedrovirus disease-resistant silkworm varieties

ABSTRACT Bombyx mori nucleopolyhedrovirus (BmNPV) disease is one of the most serious silkworm diseases, and it has caused great economic losses to the sericulture industry. So far, the disease has not been controlled effectively by therapeutic agents. Breeding resistant silkworm varieties breeding may be an effective way to improve resistance to BmNPV and reduce economic losses. A precise resistance-detection method will help to accelerate the breeding process. For this purpose, here we described the individual inoculation method (IIM). Details of the IIM include pathogen BmNPV preparation, mulberry leaf size, pathogen volume, rearing conditions, course of infection, and breeding conditions. Finally, a resistance comparison experiment was performed using the IIM and the traditional group inoculation method (GIM). The incidence of BmNPV infection and the within-group variance results showed that the IIM was more precise and reliable than the GIM.

Fusarium graminearum Inoculation on Wheat Head.

Fusarium graminearum, the major causal agent of Fusarium head blight (FHB), causes serious wheat yield losses and a threat to human and animal health. The main efforts to combat the disease are the research of pathogenesis mechanisms and breeding for disease resistance plants. The efficiency of these actions could be evaluated by reliable inoculation assay, which is performed by accurate and repeatable inoculation methods. Hence, a standard procedure of effective wheat inoculation should improve the accuracy of pathogenicity evaluation. Here, we present a protocol for wheat spike inoculation with fungal conidial suspensions or fungus agar discs. These methods show highly reproducibility and accuracy on wheat infection experiment in laboratory conditions.

Self-disinfecting surfaces and activity against Staphyloccocus aureus ATCC 6538 under real-life conditions.

Environmental surface contamination provides a potential reservoir for pathogens to cause infections. As such, self-disinfecting surfaces have been developed to possibly reduce exogenous transmission. Five different self-disinfecting surfaces were evaluated for activity against Staphylococcus aureus ATCC 6538 under real-life conditions using the dry inoculation method. Various antimicrobial effects were detected. However, following disinfection with alcoholic wipes, these effects disappeared. Further development is necessary to produce self-disinfecting surfaces that are stable in the presence of hospital disinfectants, as it is impossible to guarantee that self-disinfecting surfaces in healthcare settings will not be exposed to disinfectants.

Maintaining unperturbed cerebral blood flow is key in the study of brain metastasis and its interactions with stress and inflammatory responses.

Blood-borne brain metastases are associated with poor prognosis, but little is known about the interplay between cerebral blood flow, surgical stress responses, and the metastatic process. The intra-carotid inoculation approach, traditionally used in animal studies, involves permanent occlusion of the common carotid artery (CCA). Herein we introduced a novel intra-carotid inoculation approach that avoids CCA ligation, namely - assisted external carotid artery inoculation (aECAi) - and compared it to the traditional approach in C57/BL6 mice, assessing cerebral blood flow; particle distribution; blood-brain barrier (BBB) integrity; stress, inflammatory and immune responses; and brain tumor retention and growth. Doppler flowmetry and two-photon imaging confirmed that only in the traditional approach regional and capillary cerebral blood flux were significantly reduced. Corticosterone and plasma IL-6 levels were higher in the traditional approach, splenic numbers of NK, CD3+, granulocytes, and dendritic cells were lower, and many of these indices were more profoundly affected by surgical stress in the traditional approach. BBB integrity was unaffected. Administration of spherical beads indicated that CCA ligation significantly limited brain distribution of injected particles, and inoculation of D122-LLC syngeneic tumor cells resulted in 10-fold lower brain tumor-cell retention in the traditional approach. Last, while most of the injected tumor cells were arrested in extra-cranial head areas, our method improved targeting of brain-tissue by 7-fold. This head versus brain distribution difference, commonly overlooked, cannot be detected using in vivo bioluminescent imaging. Overall, it is crucial to maintain unperturbed cerebral blood flow while studying brain metastasis and interactions with stress and inflammatory responses.

Effect of red cyst cell inoculation and iron(II) supplementation on autotrophic astaxanthin production by Haematococcus pluvialis under outdoor summer conditions.

The negative effect of heat stress on the autotrophic astaxanthin production by Haematococcus pluvialis has been observed during outdoor culture in summer. Under the summer conditions, the proliferation of vegetative cells was highly halted in the green stage and the inducibility in the biosynthesis of astaxanthin was partly hindered in the red stage. Herein, under outdoor summer conditions in which variations of the diurnal temperature occur, heat-stress-driven inefficient vegetative growth of H. pluvialis was highly improved by inoculating the red cyst cells; thereby, maintaining relatively moderate intracellular carotenoid levels in the green stage. Subsequently, a remarkably enhanced astaxanthin titer was successfully obtained by supplementing 50 µM iron(II) to induce the heat stress-driven Haber-Weiss reaction in the red stage. As a result, the productivity of astaxanthin in the cells cultured under summer temperature conditions (23.4-33.5 °C) using the two methods of red cell (cyst) inoculation and the iron(Fe(2+)) supplementation was increased by 147% up to 5.53 mg/L day compared with that of the cells cultured under spring temperature conditions (17.5-27.3 °C). Our technical solutions will definitely improve the annual natural astaxanthin productivity in H. pluvialis in locations confronted by hot summer weather, particularly in large-scale closed photobioreactor systems.