Insecticidal features displayed by the beneficial rhizobacterium Pseudomonas chlororaphis PCL1606

The biocontrol rhizobacterium Pseudomonas chlororaphis is one of the bacterial species of the P. fluorescens group where insecticide fit genes have been found. Fit toxin, supported with other antimicrobial compounds, gives the bacterial the ability to repel and to fight against eukaryotic organisms, such as nematodes and insect larvae, thus protecting the plant host and itself. Pseudomonas chlororaphis PCL1606 is an antagonistic rhizobacterium isolated from avocado roots and show efficient biocontrol against fungal soil-borne disease. The main antimicrobial compound produced by P. chlororaphis PCL606 is 2-hexyl-5-propyl resorcinol (HPR), which plays a crucial role in effective biocontrol against fungal pathogens. Further analysis of the P. chlororaphis PCL1606 genome showed the presence of hydrogen cyanide (HCN), pyrrolnitrin (PRN), and homologous fit genes. To test the insecticidal activity and to determine the bases for such activity, single and double mutants on the biosynthetic genes of these four compounds were tested in a Galleria mellonella larval model using inoculation by injection. The results revealed that Fit toxin and HPR in combination are involved in the insecticide phenotype of P. chlororaphis PCL1606, and additional compounds such as HCN and PRN could be considered supporting compounds.

     

Detection and Location of Seed-Borne Inoculum of Didymella bryoniae and its Transmission in Seedlings of Cucumber and Pumpkin

Of the 91 tested cucurbit seed samples from thirteen countries nine from four countries were found to be infected with Didymella bryoniae. The pathogen is located on and in the seed coat including the perisperm and in the tissue of the cotyledons. Primary seedling infection occurred on the radicle, hypoctyl and cotyledons. Infection of the radicle generally caused pre-emergence tot while infection on the hypocotyl and cotyledons developed furthere inoculum for infection of the first true leaves and the stem. Experimentally, all the isolates of D. bryoniae could infect cucumber (Cucumis sativus), oriental melon (Cucumis melo var. makuwa), pumpkin (Cucurbita pepo) and watermelon (Citrullus vulgaris) at different growth stages; the susceptibility of cucumber and pumpkin was markedly influenced by previailing humid conditions.The blotter method was found more suitable for detection of seed-borne infection than the agar plate method.     

Phylogenetically closely related pseudomonads isolated from arthropods exhibit differential insect-killing abilities and genetic variations in insecticidal factors

Strains belonging to the Pseudomonas protegens and Pseudomonas chlororaphis species are able to control soilborne plant pathogens and to kill pest insects by producing virulence factors such as toxins, chitinases, antimicrobials or two-partner secretion systems. Most insecticidal Pseudomonas described so far were isolated from roots or soil. It is unknown whether these bacteria naturally occur in arthropods and how they interact with them. Therefore, we isolated P. protegens and P. chlororaphis from various healthy insects and myriapods, roots and soil collected in an agricultural field and a neighbouring grassland. The isolates were compared for insect killing, pathogen suppression and host colonization abilities. Our results indicate that neither the origin of isolation nor the phylogenetic position mirror the degree of insecticidal activity. Pseudomonas protegens strains appeared homogeneous regarding phylogeny, biocontrol and insecticidal capabilities, whereas P. chlororaphis strains were phylogenetically and phenotypically more heterogenous. A phenotypic and genomic analysis of five closely related P. chlororaphis isolates displaying varying levels of insecticidal activity revealed variations in genes encoding insecticidal factors that may account for the reduced insecticidal activity of certain isolates. Our findings point towards an adaption to insects within closely related pseudomonads and contribute to understand the ecology of insecticidal Pseudomonas.     

Isolation screening and characterisation of local beneficial rhizobacteria based upon their ability to suppress the growth of Fusarium oxysporum f. sp. radicis-lycopersici and tomato foot and root rot

Local beneficial rhizobacteria were selected based upon their ability to control the fungus Fusarium oxysporum f. sp. radicis-lycopersici which causes crown and root rot of tomato. Seven out of 384 strains prevailed in multiple and dual cultures and were identified as Pseudomonas chlororaphis (one strain), Bacillus cereus (one strain), Serratia marcescens (three strains) and Serratia rubidaea (two strains), by sequencing the 16S rRNA or the 16S and 23S rRNA inter-spacer region. The seven selected rhizobacteria were tested for their biocontrol and growth-promoting effects in planta, and their antifungal properties in vitro. All strains significantly reduced disease severity under controlled conditions, in a gnotobiotic system and in pots. Moreover, one P. chlororaphis and one S. marcescens strain significantly decreased disease severity to the level of the healthy control under natural conditions in pots experiments. The inhibitory activity of bacterial liquid cultures' metabolites on the fungus was demonstrated for all strains in vitro, using filter paper, thin layer chromatography and microtiter bioassays. Genes encoding phenazines were tentatively detected by PCR in the P. chlororaphis strain and chitinase-encoding genes were detected in one S. rubidaea and all three S. marcescens strains. Production of phenazine-1-carboxamide and hydrogen cyanide was evidenced for the P. chlororaphis strain while protease activity and production of siderophore-like compounds was confirmed in all bacterial strains. Possible use of these strains as biological control agents and their impact on natural biocontrol of pathogens in soils is discussed.     

绿针假单胞菌的研究进展及农业应用潜力

绿针假单胞菌(Pseudomonas chlororaphis)是目前研究较多的生防菌种之一.19世纪初被Miguela首次分离,将其鉴定为假单胞菌(Pseudomonas),并将机会性病原菌绿脓杆菌作为其模式菌株,而后Peix于2007年重新将其分类为绿针假单胞菌(P.chlororaphis).目前该菌种已报道有4...     

Host Specificity and Genetic Diversity of Didymella bryoniae from Cucurbitaceae in Brazil

Thirty‐seven isolates of Didymella bryoniae from three Cucurbitaceae species were collected in Brazil and tested for pathogenicity to watermelon. All isolates were pathogenic but differed in aggressiveness levels. Seven representative isolates were used in cross‐pathogenicity tests against 10 cucurbitaceous hosts. Most isolates were pathogenic to most host species tested, except to Sechium edule. Among the susceptible species, Citrullus and Cucumis species were the most susceptible hosts, while pumpkin and Luffa purgans were the most resistant. Host of origin affected the pattern of aggressiveness on each host. Isolates from watermelon were very aggressive to their original host, but much less aggressive or not pathogenic at all to some Cucurbita. Two previously described random‐amplified polymorphic DNA (RAPD)‐specific primers indicated that 81% of the isolates could be classified into the so‐called RG I group, while the remaining isolates could not be classified into any of the described RG groups. All 37 isolates were further characterized by RAPD fingerprinting and compared with three US isolates representative of RG I and RG II groups. The Brazilian D. bryoniae isolates could be separated into genetically similar clusters. The majority of the isolates were grouped in cluster DB Ia, which contained only isolates of Citrullus lanatus and Cucumis melo. Two of the American isolates used as controls clustered with this group at 68% similarity level. The DB Ib cluster included three Brazilian isolates obtained from melon and watermelon and the American representative for RG II, at a lower similarity level (43%). Two isolates from watermelon clustered with one isolate from melon in a separate group (DB II), while one single isolate from pumpkin (DB III) showed the lowest genetic similarity to all other isolates. Didymella bryoniae isolates from Brazil showed, therefore, a level of genetic diversity higher than previously reported for the species. RAPD fingerprinting allowed for geographical distinction of D. bryoniae isolates but no correlation between genetic distance, aggressiveness or origin of the isolate was found.     

Characterisation of Pseudomonas chlororaphis subsp. aurantiaca strain Pa40 with the ability to control wheat sharp eyespot disease

This study details the isolation and characterisation of Pseudomonas chlororaphis subsp. aurantiaca strain Pa40, and is the first to examine P. chlororaphis for use in suppression of wheat sharp eyespot on wheat. Pa40 was isolated during an investigation aimed to identify biocontrol agents for Rhizoctonia cerealis. Over 500 bacterial strains were isolated from the rhizosphere of infected wheat and screened for in vitro antibiosis towards R. cerealis and ability to provide biocontrol in planta. Twenty‐six isolates showed highly antagonistic activity towards R. cerealis, in which Pseudomonas spp. and Bacillus spp. were predominant members of the antagonistic community. Strain Pa40 exhibited clear and consistent suppression of wheat sharp eyespot disease in a greenhouse study and suppression was comparable to that of chemical treatment with validamycin A. Pa40 was identified as P. chlororaphis subsp. aurantiaca by the Biolog identification system combined with 16S rDNA, atpD, carA and recA sequence analysis and biochemical and physiological characteristics. To determine broad‐spectrum applicability and the specific mechanisms involved in Pa40's pathogen suppression this strain was tested for antibiosis towards various phytopathogens and assayed for many biocontrol activities and plant‐beneficial traits. Strain Pa40 inhibited the growth of 10 of 13 phytopathogenic fungal strains and six of eight phytopathogenic bacteria tested. This original work characterises HCN, protease and siderophore production in P. chlororaphis. Each of these characteristics likely contributed to Pa40's biocontrol capabilities as well as stimulation of the hypersensitive response in tobacco and the presence of genes involved in the biosynthesis of phenazine, 2‐hydroxylated phenazine and pyrrolnitrin.     

Pseudomonads: major antagonistic endophytic bacteria to suppress bacterial wilt pathogen, <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in the eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Endophytic bacteria of eggplant, cucumber and groundnut were isolated from different locations of Goa, India. Based on in vitro screening, 28 bacterial isolates which effectively inhibited Ralstonia solanacearum, a bacterial wilt pathogen of the eggplant were characterized and identified. More than 50% of these isolates were Pseudomonas fluorescens in which a vast degree of variability was found to exist when biochemical characteristics were compared. In greenhouse experiments, the plants treated with Pseudomonas isolates (EB9, EB67), Enterobacter isolates (EB44, EB89) and Bacillus isolates (EC4, EC13) reduced the wilt incidence by more than 70%. All the selected isolates reduced damping off by more than 50% and improved the growth of seedlings in the nursery stage. Most of the selected antagonists produced an antibiotic, DAPG, which inhibited R. solanacearum under in vitro conditions and might have been responsible for reduced wilt incidence under in vivo conditions. Also production of siderophores and IAA in the culture medium by the antagonists was recorded, which could be involved in biocontrol and growth promotion in crop plants. From our study we conclude that Pseudomonas is the major antagonistic endophytic bacteria from eggplants which have the potential to be used as a biocontrol agent as well as plant growth-promoting rhizobacteria. Large scale field evaluation and detailed knowledge on antagonistic mechanism could provide an effective biocontrol solution for bacterial wilt of solanaceous crops.     

Quorum quenching by Bacillus cereus U92: a double-edged sword in biological control of plant diseases

In the present survey, quorum quenching activity was examined from a biocontrol point of view. Acyl-homoserine lactone (AHL) degrading bacteria were isolated from tomato rhizosphere using two standard bioreporter strains and different synthetic AHLs and then identified according to 16S rDNA sequences. Five isolates capable of inactivating both short and long 3oxo-substituted AHLs showed high similarity with the genera Bacillus, Microbacterium and Arthrobacter, and thereby Bacillus cereus U92 was determined as the most efficient quorum quencher strain. In the quantitative experiments, this strain remarkably inactivated all synthetic AHLs up to 80%. In the laboratory co-cultures, B. cereus U92 efficiently quenched QS-regulated phenotypes in Agrobacterium tumefaciens, Pseudomonas aeruginosa, Pseudomonas chlororaphis and Chromobacterium violaceum. The strain successfully reduced the frequency of Ti-plasmid conjugal transfer in A. tumefaciens by about 99% in the binary cultures. Meanwhile, in a more natural environment, this strain acted as a biocontrol agent, efficient in alleviating QS-regulated crown gall incidence on tomato roots (up to 90%) as well as attenuating Pectobacterium soft rot on potato tubers (up to 60%). On the other hand, reducing phenazine production in P. chlororaphis operated as a suppressor of its QS-regulated biocontrol activity and also inhibited pyocyanin production in P. aeruginosa, a plant growth-promoting bacterium, by 75%. In general, B. cereus U92 seems very promising in the biological control of pathogenic bacteria; however, its broad AHL-degrading activity has a detrimental role on beneficial microbes which should not be neglected.     

Interactions of Colletotrichum musae and Lasiodiplodia theobromae and their biocontrol by Pantoea agglomerans and Flavobacterium sp. in expression of crown rot of “Embul” banana

Previous research has shown that two local isolates of bacteria (Pantoea agglomerans and Flavobacterium sp.) are capable of biocontrol of the two main pathogens (Colletotrichum musae and Lasiodiplodia theobromae) known to cause crown rot on “Embul” (Musa, AAB) banana. In this investigation an attempt was made to elucidate the comparative virulence of these pathogens and to determine the underlying biocontrol mechanisms. L. theobromae was more virulent, causing faster spread of the disease, whereas C. musae was more resistant to the bacterial antagonists. Viable cells of the antagonists were more effective at suppressing conidial germination than cell-free culture media. It seemed that antifungal compounds acting on conidial germination may be heat stable and those acting on mycelia may include heat labile compounds also. Considering the specialized roles observed for each pathogen in terms of causing the disease, and in modes of control by antagonists, future field investigations on biocontrol should consider the roles played by the pathogens and the antagonists.

Production of Rhamnolipids by Pseudomonas chlororaphis, a Nonpathogenic Bacterium

Rhamnolipids, naturally occurring biosurfactants constructed of rhamnose sugar molecules and β-hydroxyalkanoic acids, have a wide range of potential commercial applications. In the course of a survey of 33 different bacterial isolates, we have identified, using a phenotypic assay for rhamnolipid production, a strain of the nonpathogenic bacterial species Pseudomonas chlororaphis that is capable of producing rhamnolipids. Rhamnolipid production by P. chlororaphis was achieved by growth at room temperature in static cultures of a mineral salts medium containing 2% glucose. We obtained yields of roughly 1 g/liter of rhamnolipids, an amount comparable to the production levels reported in Pseudomonas aeruginosa grown with glucose as the carbon source. The rhamnolipids produced by P. chlororaphis appear to be exclusively the mono-rhamnolipid form. The most prevalent molecular species had one monounsaturated hydroxy fatty acid of 12 carbons and one saturated hydroxy fatty acid of 10 carbons. P. chlororaphis, a nonpathogenic saprophyte of the soil, is currently employed as a biocontrol agent against certain types of plant fungal diseases. The pathogenic nature of all bacteria previously known to produce rhamnolipids has been a major obstacle to commercial production of rhamnolipids. The use of P. chlororaphis therefore greatly simplifies this matter by removing the need for containment systems and stringent separation processes in the production of rhamnolipids.     

Biocontrol Potential of Soybean Bacterial Endophytes Against Charcoal Rot Fungus, <Emphasis Type="Italic">Rhizoctonia bataticola</Emphasis>

A total of 137 bacterial isolates from surface sterilized root, stem, and nodule tissues of soybean were screened for their antifungal activity against major phytopathogens like Rhizoctonia bataticola, Macrophomina phaseolina, Fusarium udam, and Sclerotium rolfsii. Nine bacterial endophytes suppressed the pathogens under in vitro plate assay. These were characterized biochemically and identified at the genus level based on their partial sequence analysis of 16S rDNA. Eight of the isolates belonged to Bacillus and one to Paenibacillus. The phylogenetic relationship among the selected isolates was studied and phylogenetic trees were generated. The selected isolates were screened for biocontrol traits like production of hydrogen cyanide (HCN), siderophore, hydrolytic enzymes, antibiotics, and plant growth promoting traits like indole 3-acetic acid production, phosphate solubilization, and nitrogen fixation. A modified assessment scheme was used to select the most efficient biocontrol isolates Paenibacillus sp. HKA-15 (HKA-15) and Bacillus sp. HKA-121 (HKA-121) as potential candidates for charcoal rot biocontrol as well as soybean plant growth promotion.     

Production of the antifungal compounds phenazine and pyrrolnitrin from Pseudomonas chlororaphis O6 is differentially regulated by glucose

Aims: To determine whether glucose in growth medium affects secondary metabolite production and biocontrol efficacy of Pseudomonas chlororaphis O6. Methods and Results: The secondary metabolites pyrrolnitrin and phenazines antagonize phytopathogenic fungi. The expression of the prnA gene encoding tryptophan halogenase, the first step in pyrrolnitrin biosynthesis, required the stationary‐phase sigma factor, RpoS. Mutations in rpoS and prnA in Ps. chlororaphis O6 eliminated antifungal activity against Rhizoctonia solani and Fusarium graminearum. Pyrrolnitrin production was reduced by glucose in growth media, whereas phenazine levels were increased. The efficacy of Ps. chlororaphis O6 in the biocontrol of tomato late blight was reduced by addition of glucose to the growth medium. Conclusions: Regulation by glucose of pyrrolnitrin production influenced the efficacy of the biocontrol of tomato leaf blight. Significance and Impact of the Study: The nutritional regulation of secondary metabolite production from a soil pseudomonad may account, at least in part, for the variability of biocontrol under field conditions.     

Biocontrol potential of five Burkholderia and Pseudomonas strains against Colletotrichum truncatum infecting chilli pepper

The effect of bacterial antagonism against the causal agent, Colletotrichum truncatum was assessed as a potential alternative in managing anthracnose in chilli pepper. Out of 104 contrasting bacterial colonies isolated from rhizosphere soil of a forest floor, five isolates caused the radial growth inhibition greater than 90% (Significant at p?<?0.05 level) of C. truncatum in dual cultures. Based on 16S rRNA analysis, these antagonistic bacterial isolates were identified as Pseudomonas aeruginosa, Burkholderia arboris, Burkholderia gladioli and Burkholderia rinojensis. The selected promising antagonists showed nearly 100% inhibition of the spore germination of C. truncatum in vitro. These antagonists produced antifungal compounds which are diffusible in nature. Microscopic studies of blackened fungal hyphae, which were subjected to antagonism showed many deformations such as thickening, swelling and malformation. In vivo study revealed that C. truncatum inoculated chilli pepper seeds treated with the five antagonists significantly inhibited the incidence of seed colonisation (p?<?0.05) by the pathogen. At the post emergence stage, the survival percentages and vigour indices of all the antagonists’ treatments, except B. rinojensis strain 1, were significantly higher compared to the untreated control. The efficacy of the selected antagonists in managing anthracnose fruit rot was 100% at the colour breaking stage of chilli pepper fruits. These bacterial antagonists had a negative effect on C. truncatum spore attachment and subsequent colonisation on chilli pepper leaves except in the treatment of B. arboris. The results of in-vitro and in-vivo studies, suggest that the screened antagonistic bacterial isolates are potential biocontrol agents and need to be further studied for the biochemical basis of their activity against C. truncatum.     

Molecular and phenotypic characterization of potential plant growth-promoting <Emphasis Type="Italic">Pseudomonas</Emphasis> from rice and maize rhizospheres

In this study, Pseudomonas species were isolated from the rhizospheres of two plant hosts: rice (Oryza sativa cultivar Pathum Thani 1) and maize (Zea mays cultivar DK888). The genotypic diversity of isolates was determined on basis of amplified rDNA restriction analysis (ARDRA). This analysis showed that both plant varieties selected for two distinct populations of Pseudomonas. The actual biocontrol and plant promotion abilities of these strains was confirmed by bioassays on fungal (Verticillum sp., Rhizoctonia solani and Fusarium sp.) and bacterial (Ralstonia solanacearum and Bacillus subtilis) plant pathogens, as well as indole-3-acetic acid (IAA) production and carbon source utilization. There was a significant difference between isolates from rice and maize rhizosphere in terms of biological control against R.  solanacearum and B.  subtilis. Interestingly, none of the pseudomonads isolated from maize rhizosphere showed antagonistic activity against R.  solanacearum. This study indicated that the percentage of pseudomonad isolates obtained from rice rhizosphere which showed the ability to produce fluorescent pigments was almost threefold higher than pseudomonad isolates obtained from maize rhizosphere. Furthermore, the biocontrol assay results indicated that pseudomonad isolated from rice showed a higher ability to control bacterial and fungal root pathogens than pseudomonad isolates obtained from maize. This work clearly identified a number of isolates with potential for use as plant growth-promoting and biocontrol agents on rice and maize.     

Identity of plant,lichen and moss species connects with microbial abundance and soil functioning in maritime Antarctica

Background and aims

Plant breeding activities shape the rhizosphere microbiome but less is known about the relationship of both with the seed microbiome. We analyzed the composition of bacterial communities of seeds and rhizospheres of Styrian oil pumpkin genotypes in comparison to bulk soil to elucidate specific microbial signatures to support a concept involving plant-microbe interactions in breeding strategies.

Methods

The seed and rhizosphere microbiomes of 14 genotypes of oilseed pumpkin and relatives were analyzed using a 16S rRNA gene amplicon sequencing approach, which was assessed by bioinformatics and statistical methods.

Results

All analyzed microhabitats were characterized by diverse bacterial communities, but the relative proportions of phyla and the overall diversity was different. Seed microbiomes were characterized by the lowest diversity and dominant members of Enterobacteriaceae including potential pathogens (Erwinia, Pectobacterium). Potential plant-beneficial bacteria like Lysobacter, Paenibacillus and Lactococcus contributed to the microbial communities in significant abundances. Interestingly, strong genotype-specific microbiomes were detected for seeds but not for the rhizospheres.

Conclusions

Our study indicates a strong impact of the Cucurbita pepo genotype on the composition of the seed microbiome. This should be considered in breeding of new cultivars that are more capable of exploiting beneficial indigenous microbial communities.

     

In vitro Antagonism of Rhizobacteria Isolated from Coffea arabica L. against Emerging Fungal Coffee Pathogens

In vitro antagonistic effects of rhizobacteria associated with Coffea arabica L. against some fungal coffee pathogens were studied. The aims were to screen indigenous coffee‐associated isolates for their inherent antagonistic potential against major coffee wilt diseases induced by Fusarium spp. Antagonistic effects, siderophore, HCN and lytic enzyme production were determined on standard solid media. Chemical methods were employed to categorize the major types of siderophores. From a total of 212 rhizobacterial isolates tested, over 10 % (all Pseudomonas and Bacillus spp.) exhibited remarkable inhibition against Fusarium spp. One isolate AUPB24 (P. chlororaphis) showed maximum inhibition of mycelial growth against all fungal pathogens tested, whereas other isolates were mostly inhibitory to F. stilboides and F. oxysporum. The isolate AUBB20 (B. subtilis) was most antagonistic to F. xylarioides. Of the rhizobacterial isolates tested, 67 % produced siderophores and 35 % produced HCN. Many strains (all Pseudomonas spp.) produced siderophores of the hydroxamate type and only a small proportion produced those of the catecholate type. Few antagonists showed chitinase activity. The production of siderophores and HCN by Pseudomonas spp., lipase and protease by all antagonists and β‐1,3‐glucanase by several Bacillus spp. could be considered the major mechanisms involved in the inhibition of fungal growth. The in vitro results provide the first evidence of an antagonistic effect of coffee‐associated rhizobacteria against the emerging fungal coffee pathogens F. stilboides and F. xylarioides and indicate the potential of both bacterial groups for biological control of coffee wilt diseases.     

Phage biocontrol to combat Pseudomonas syringae pathogens causing disease in cherry

Bacterial canker is a major disease of Prunus species, such as cherry (Prunus avium). It is caused by Pseudomonas syringae pathovars, including P. syringae pv. syringae (Pss) and P. syringae pv. morsprunorum race 1 (Psm1) and race 2 (Psm2). Concerns over the environmental impact of, and the development of bacterial resistance to, traditional copper controls calls for new approaches to disease management. Bacteriophage-based biocontrol could provide a sustainable and natural alternative approach to combat bacterial pathogens. Therefore, seventy phages were isolated from soil, leaf and bark of cherry trees in six locations in the south east of England. Subsequently, their host range was assessed against strains of Pss, Psm1 and Psm2. While these phages lysed different Pss, Psm and some other P. syringae pathovar isolates, they did not infect beneficial bacteria such as Pseudomonas fluorescens. A subset of thirteen phages were further characterized by genome sequencing, revealing five distinct clades in which the phages could be clustered. No known toxins or lysogeny-associated genes could be identified. Using bioassays, selected phages could effectively reduce disease progression in vivo, both individually and in cocktails, reinforcing their potential as biocontrol agents in agriculture.     

Physiological stage drives fungal community dynamics and diversity in Leptospermum scoparium (mānuka) flowers

Flowers are an important niche for microbes, and microbes in turn influence plant fitness. As flower morphology and biology change rapidly over time, dynamic niches for microbes are formed and lost. Floral physiology at each life stage can therefore influence arrival, persistence and loss of microbial species; however, this remains little understood despite its potential consequences for host reproductive success. Through internal transcribed spacer 1 (ITS1) community profiling, we characterized the effect of transitioning through five floral stages of mānuka (Leptospermum scoparium), from immature bud to spent flower, and subsequent allocation to seed, on the flower-inhabiting fungal community. We found nectar-consuming yeasts from Aureobasidium and Vishniacozyma genera and functionally diverse filamentous fungi from the Cladosporium genus dominated the anthosphere. The candidate core microbiota persisted across this dynamic niche despite high microbial turnover, as observed in shifts in community composition and diversity as flowers matured and senesced. The results demonstrated that floral stages are strong drivers of anthosphere fungal community assembly and dynamics. This study represents the first detailed exploration of fungi through floral development, building on fundamental knowledge in microbial ecology of healthy flowers.