Visual and PCR assessment of light leaf spot (Pyrenopeziza brassicae) on winter oilseed rape (Brassica napus) cultivars

Methods to assess light leaf spot ( Pyrenopeziza brassicae ) on winter oilseed rape cultivars were compared in laboratory, controlled-environment and field experiments. In controlled-environment experiments with seedling leaves inoculated at GS 1,4, the greatest differences in percentage area affected by P. brassicae sporulation were observed with inoculum concentrations of 4 × 10³ or 4 × 10⁴ spores mL⁻¹, rather than 4 × 10² or 4 × 10⁵ spores mL⁻¹, but older leaves had begun to senesce before assessment, particularly where they were severely affected by P. brassicae . In winter oilseed rape field experiments, a severe light leaf spot epidemic developed in 2002/03 (inoculated, September/October rainfall 127·2 mm) but not in 2003/04 (uninoculated, September/October rainfall 40·7 mm). In-plot assessments discriminated between cultivars best in February/March in 2003 and June in 2004, but sometimes failed to detect plots with many infected plants (e.g. March/April 2004). Ranking of cultivar resistance differed between seedling experiments done under controlled-environment conditions and field experiments. The sensitivity of detection of P. brassicae DNA extracted from culture was greater using the PCR primer pair PbITSF/PbITSR than using primers Pb1/Pb2. P. brassicae was detected by PCR (PbITS primers) in leaves from controlled-environment experiments immediately and up to 14 days after inoculation, and in leaves sampled from field experiments 2 months before detection by visual assessment.     

Efficient, long-lasting resistance against the soft rot bacterium Pectobacterium carotovorum in calla lily provided by the plant activator methyl jasmonate

The potential of three externally applied chemical plant activators, Bion, BABA and methyl jasmonate, known to act only through the plant defence system and not on the pathogen directly, to induce resistance against wild-type Pectobacterium carotovorum was examined in white-flowered calla lily ( Zantedeschia aethiopica ). Following a 24-h induction period, plants were challenge-inoculated with P. carotovorum , originally isolated from calla lily or potato plants, previously transformed using a gfp broad-host-range promoter-probe vector. After another 24 h, Bion treatment (10  µ g mL⁻¹, as a drench) reduced disease symptoms more than sixfold and bacterial proliferation by four orders of magnitude. BABA treatment (5–10  µ g mL⁻¹, also as a drench) reduced the rate of infection by 75–85%. However, the protection afforded by both inducers did not persist. Also, at higher concentrations both displayed a phytotoxic effect. By contrast, methyl jasmonate (10 m m , applied as a leaf spray) completely inhibited P. carotovorum development in calla lily leaves and afforded a long-lasting effect. It is suggested that the defence response of calla lily against P. carotovorum involves the SA-signalling pathway in the short term, but the jasmonate/ethylene-signalling pathway is required for durable protection.     

Berry infection and the development of bunch rot in grapes caused by Aspergillus carbonarius

The effects of different levels of inoculum of Aspergillus carbonarius and time of inoculation on berry infection and the development of aspergillus bunch rot on grapevines (cv. Sultana) were studied under field conditions. Inflorescences at full bloom were inoculated with aqueous spore suspensions of A. carbonarius containing 0 or 1 × 10⁶ spores mL⁻¹ in 2004/05 and 0, 1 × 10² or 1 × 10⁵ spores mL⁻¹ in 2005/06. In both years, the incidence of infection in inoculated berries was significantly higher than in uninoculated berries. Incidence of infection in berries from veraison until harvest was higher than at earlier stages of bunch development (berry set to berries that were still hard and green). Inoculation of bunches at veraison did not significantly increase A. carbonarius infection prior to harvest, at harvest, 6 days after harvest or when berries were over-ripe. Bunches inoculated at harvest did not significantly increase infection 6 days after harvest or when berries were over-ripe. Aspergillus carbonarius was isolated more frequently from the pedicel end (53·1%) than from the middle section (37·5%) and distal end (35·0%) of berries that were inoculated with 10⁵ spores mL⁻¹.     

Characterization and differentiation of soft rot causing Pectobacterium carotovorum of Indian origin

Twenty different strains of Pectobacterium carotovorum (Pcc) were recovered from vegetable-growing fields of Vadodara, Gujarat (India) using a plant host enrichment approach during the years 2006–9. The isolated strains, based on differences in physiological and biochemical features, were classified into five different biovars, and then identified as Pectobacterium carotovorum (Pcc) by species-specific PCR and 16S rDNA sequences. Moreover, these Pcc strains were also differentiated based on virulence traits, plant cell wall degrading enzymes production and phylogenetic analysis of 16S rDNA sequence and Repetitive extragenic palindromic—PCR (rep-PCR). High genetic variability, independent of their pathogenicity, was revealed among these Pcc strains by rep-PCR typing using four different primer sets . Factors other than the plant host specificity seem to correlate with genetic variability of these economically important Pcc strains. Significantly, polyphasic characterization of the Pcc strains clearly reveals the heterogeneity among them. The present studies can be considered as useful epidemiological surveillance (or distribution) of soft rot causing Pcc in the semi arid region of India.     

Antifungal vapour-phase activity of allyl-isothiocyanate against Penicillium expansum on pears

The fungitoxicity of allyl-isothiocyanate (AITC) vapour against Penicillium expansum , the causal agent of blue mould on pears (cvs Conference and Kaiser), was evaluated. The best control of blue mould was obtained by exposing fruits for 24 h in a 5 mg L⁻¹ AITC-enriched atmosphere, the extent of control depending on the inoculum density. Lesion diameter was inversely related to AITC concentration. In treated fruits the percentage of infected wounds increased with conidial concentration, with fewer than 20% affected at 1 × 10³ conidia mL⁻¹ to almost 80% at 1 × 10⁶ conidia mL⁻¹. In comparison, >98% of wounds were infected in untreated fruits irrespective of conidial concentration. AITC treatments were effective up to 24 h after inoculation for Conference and 48 h for Kaiser. AITC treatments also controlled a thiabendazole-resistant strain of P. expansum , reducing the incidence of blue mould by 90% in both cultivars. The use of AITC produced from pure sinigrin or from Brassica juncea defatted meal may be an economically viable alternative to synthetic fungicides against P. expansum .     

Effects of avirulent bacteriocin-producing strains of Pseudomonas solanacearum on the control of bacterial wilt of tobacco

Root systems of tobacco dipped in suspensions containing 2 × 10⁹ colony forming units (CFU)/ml of avirulent bacteriocin-producing strains (ABPS) of Pseudomonas solanacearum and assayed immediately after planting in steam-sterilized soil had 8 × 10⁶ CFU/root system of ABPS. The bacterial population declined to an average of 5·3 × 10⁵ CFU/root system after 30 days. Roots of seedlings dipped in bacterial suspensions of ABPS were more effectively protected against wilt caused by P. solanacearum than those dipped in suspensions of an avirulent nonbacteriocin-producing strain (ANBPS). Lipopolysaccharide (LPS) isolated from one ABPS (121) inhibited the attachment of bacteria on roots by 70% but had no effect on the reduction of wilt, whereas bacterial cells significantly reduced the disease severity as compared to LPS or water treatment. In steam-sterilized soil containing a 1:1 mixture (5 × 10⁵ CFU/g of oven-dried soil) of ABPS 121 or 237 and the virulent strain K-60, ABPS 121 reduced multiplication of the virulent strain in soil and in the rhizosphere of seedlings. When roots of seedlings were dipped in a suspension of 2 × 10⁹ CFU/ml of ABPS before planting, root colonization by the virulent strain added to steam-sterilized soil at 2 × 10⁶ CFU/g of oven-dried soil was significantly reduced. When roots were dipped in a suspension of ABPS and assayed 20 days after planting, 98% of the bacterial population was found in the original zone of inoculation and only 2% was detected in new growths of the root system. Plants which were grown in soil infested with ABPS 121 or K-60 had both strains present at variable populations along all sections of roots.     

Detection of Xylophilus ampelinus in grapevine cuttings using a nested polymerase chain reaction

A sensitive and specific assay was developed to detect bacterial blight of grapevine caused by Xylophilus ampelinus (Panagopoulos, 1969) comb. nov. in grapevine cuttings. The 16S−23S rDNA intergenic spacer region of X. ampelinus was sequenced and pathogen-specific primers were designed from a region in the spacer between the tRNA (Ala) and the 23S genes. A nested PCR (n-PCR) reaction was applied with a first-stage PCR using universal primers within the ends of the 16S and 23S genes, followed by a second-stage PCR with nested primers specific to the X. ampelinus spacer region. A 277-bp fragment was amplified from 38 Xylophilus strains tested, but not from saprophytes associated with grapevine or phylogenetically related phytobacteria. The 277-bp product was shown to be derived from the X. ampelinus spacer region by restriction with Dra I, Sau 3AI, Taq I and Msp I, Southern hybridization and genomic DNA dot blots. When the (n-PCR) procedure was applied in the absence of nontarget DNA, the limit of detection was less than 10 colony-forming units (CFU) per µ L. The same number of  X. ampelinus CFU could be detected in the presence of 1·5 × 10⁵ CFU  µ L⁻¹ of Erwinia herbicola cells using the n-PCR procedure.     

Factors influencing infection of mechanical wounds by Fusarium circinatum on Monterey pines (Pinus radiata)

Pitch canker, caused by the fungus Fusarium circinatum , is a disease affecting many pine tree species. In California, Pinus radiata (Monterey pine) is the principal pine host affected by pitch canker. This investigation into factors affecting infection frequency by F. circinatum of P. radiata examined the influence of: (i) wound size; (ii) relative humidity; (iii) time of inoculation; (iv) inoculum density; and (v) wound age. Wounded branches sustained significantly more infections when large-diameter (1·6 mm) rather than small-diameter (0·5 mm) wounds were made. Infection frequencies tended to be higher at 100% RH than at ambient humidity, although these differences were not statistically significant. Infection frequencies were significantly higher on branches inoculated after 17·00 h than on branches inoculated before noon. Infection frequencies were significantly higher for wounded branches spray-inoculated with 5 × 10⁷ rather than 1 × 10⁷ spores mL⁻¹. Infection frequencies of pruning wounds declined as wounds aged.     

Effects of calcium on biocontrol activity of yeast antagonists against the postharvest fungal pathogen Rhizopus stolonifer

Calcium chloride (2% w/v) significantly inhibited the growth of the pathogen Rhizopus stolonifer , but did not affect the colony-forming units (CFU) of yeasts Candida guilliermondii and Pichia membranefaciens in potato dextrose broth. The concentration of yeast suspension influenced spore germination and germ tube growth of R. stolonifer in vitro, as well as disease incidence and lesion development in fruits. There were significant negative relationships between the suspension concentrations of the yeasts and the growth as well as infectivity of the pathogen. The addition of calcium resulted in lower spore germination rates and slower growth of germ tubes in vitro , as well as in lower disease incidences and smaller lesion diameters compared with treatments with yeast antagonists alone. When yeast cell suspensions reached a concentration of 5 × 10⁸ CFU mL⁻¹, growth of the pathogen was completely limited in vitro , and no infection was found in peach and nectarine fruits treated with or without calcium.     

Interaction of a bioherbicide and glyphosate for controlling hemp sesbania in glyphosate-resistant soybean

The bioherbicidal fungus, Colletotrichum truncatum (Schwein.) Andrus & Moore, was tested at different inoculum concentrations alone and in combination with, prior to or following treatment with different rates of glyphosate ( N -[phosphonomethyl]glycine) (Roundup Ultra) for the control of hemp sesbania ( Sesbania exaltata [Raf.] Rydb. ex A.W. Hill) in Roundup Ready soybean field plots. Colletotrichum truncatum and glyphosate were applied in all pair-wise combinations of 0, 1.25, 2.5, 5.0, and 10.0 × 10⁶ spores mL⁻¹ (i.e. 3.125, 6.25, 12.5, and 25 × 10¹¹ spores ha⁻¹), and 0.15, 0.30, 0.60, and 1.2 kg ha⁻¹, respectively. Weed control and disease incidence were enhanced at the two lowest fungal and herbicidal rates when the fungal spores were applied after glyphosate treatment. The application of the fungus in combination with or prior to glyphosate application at 0.30 kg ha⁻¹ resulted in reduced disease incidence and weed control regardless of the inoculum's concentration. At the highest glyphosate rates, the weeds were controlled by the herbicide alone. These results suggest that it might be possible to utilize additive or synergistic herbicide and pathogen interactions to enhance hemp sesbania control.     

Factors influencing infection of eucalypts by Cylindrocladium pteridis

The pattern of Cylindrocladium pteridis adhesion, germination and penetration in eucalypt leaves was assessed using scanning electron microscopy. The effects of inoculum concentration, leaf wetness period, plant age and branch position of cylindrocladium leaf blight and defoliation severity were assessed in greenhouse studies using two Eucalyptus grandis × E. urophylla hybrid clones. Penetration occurred through stomata, and there was no difference in the number of penetrations between young and old leaves. Percentage leaf area with lesions and defoliation increased with the increase in inoculum concentration (1 × 10² to 10⁵ conidia mL⁻¹), duration of leaf wetness period (6 to 48 h) and plant age (60 to 180 days). Branch position in plants also significantly affected the percentage leaf area with lesions and defoliation, the latter variable being significantly higher at the stem base. The highest values of lesion area were also observed on leaves at the stem base in both clones. The Pearson correlation between defoliation and leaf area with lesions was significant in all experiments ( r  > 0·9) indicating a high association between these two variables.     

Sensitivity of Sclerotinia homoeocarpa to demethylation-inhibiting fungicides in Ontario, Canada, after a decade of use

In late 2003, nine populations of Sclerotinia homoeocarpa in Ontario Canada (seven of which had been previously sampled in early 1994, prior to the registration of sterol demethylation-inhibiting (DMI) fungicides for turf disease control in Canada) were sampled and tested for sensitivity to propiconazole. Four of the nine populations had not been treated with DMI fungicides during the intervening years, and isolates from these locations were sensitive to propiconazole (geometric mean EC₅₀ values of 0·005–0·012 µ g mL⁻¹, compared with 0·005–0·008 µ g mL⁻¹ for the original 1994 populations). Among the five populations from 2003 that had been exposed to DMI fungicides, mean EC₅₀ values were significantly greater, ranging from 0·020 to 0·048 µ g mL⁻¹. A significant correlation of determination was found between estimated number of fungicide applications and log EC₅₀ ( R ² = 0·832, P  = 0·0001), and the equation predicted that 42·3 applications of propiconazole would be needed to bring a sensitive population (EC₅₀ < 0·01  µ g mL⁻¹) to a resistant level (EC₅₀ > 0·10  µ g mL⁻¹). Fungicide sensitivity vs. duration of fungicide efficacy was also tested, and it was found that isolates with decreased sensitivity were able to more quickly overcome the inhibitory effects of fungicide application, reducing the duration of control from 3 weeks to 2 weeks.     

Identification and sensitive endophytic detection of the fire blight pathogen Erwinia amylovora with 23S ribosomal DNA sequences and the polymerase chain reaction

Two short sequences, situated in the bacterial 23S rDNA gene, were used as primers for the PCR detection of Erwinia amylovora bacteria. All 34 E. amylovora strains tested, coming from different geographical and host plant origins and of different virulence, produced a 565-bp PCR fragment. The E. amylovora bacteria could be discriminated from all other phytobacteria with which no PCR product was observed. Only Escherichia coli bacteria were cross-recognized by the production of a weaker PCR band of similar size to E. amylovora . In a fast PCR protocol, where two temperatures were cycled, E. amylovora in pure culture could be detected on gel at concentrations as low as 3 × 10² cfu mL^–1. This corresponds to a detection limit of 1.5 bacteria per PCR. However, reliable PCR detection in woody host plant tissue was only obtained with PVP/PVPP-treated sample extracts. Using E. amylovora -spiked plant extracts and extracts of fruit tree shoots artificially infected with E. amylovora , the PCR detection sensitivity was determined to be 6.6 × 10² cfu mL^–1 of extract. Starting from the plant samples, the PCR detection results were visualized on gels within 5 h.     

Differential susceptibility of barnyard grass (Echinochloa crus-galli var. crus-galli) ecotypes to Exserohilum longirostratum

Ten barnyard grass ( Echinochloa crus-galli var. crus-galli ) ecotypes collected from several locations in Malaysian and Indonesian granaries were tested for variation in their susceptibility to the leaf blight pathogen ( Exserohilum longirostratum ). Four phenological growth stages of the ecotypes were sprayed with E. longirostratum at the rate of 1 × 10⁷ conidia mL⁻¹ under glasshouse conditions. The results of the study showed that plants inoculated with 1 × 10⁷ conidia mL⁻¹ and exposed for 24 h dew periods were severely infected and the percentage dry weight reduction of all ecotypes significantly increased. The 1–2 and 2–3-leaf stage plants were completely killed within 6 days after inoculation. However, susceptibility reactions among the ecotypes were observed at the 4–5-leaf stage, while the 6–7-leaf stage was resistant. The ecotypes, K-02, PK-04, KN-02, CJ-01, and L-01, were the most susceptible ecotypes as indicated by a higher area under disease progress curve value. The apparent disease progress rates for these ecotypes were significantly faster compared to the other ecotypes. The dry weight reduction among the four growth stages was variable. These findings show that the 1–2 and 2–3-leaf stages are the most susceptible, while the 6–7-leaf stage was resistant. This study has confirmed that a variable response to the fungal pathogen occurred within an intraspecific barnyard grass collection.     

Management of late leaf spot of groundnut (Arachis hypogaea) with chlorothalonil-tolerant isolates of Pseudomonas aeruginosa

Fifteen groundnut-associated bacterial isolates that inhibited by > 90% the in vitro conidial germination of Phaeoisariopsis personata , causal agent of late leaf spot disease of groundnut, were applied as a prophylactic spray (10⁸ cfu mL⁻¹) and tested for control of the disease in the glasshouse. Two groundnut seed-associated bacterial isolates, GSE 18 and GSE 19, identified as Pseudomonas aeruginosa , reduced the lesion frequency (LF) by up to 70%. A 90-day-old peat-based formulation of P. aeruginosa GSE 18 reduced LF measured 15 days postinoculation by up to 60%. Both P. aeruginosa GSE 18 and GSE 19 were tolerant to chlorothalonil (Kavach®) up to 2000  µ g mL⁻¹ in LB broth. In glasshouse trials, GSE 18 and GSE 19 tested in combination with reduced concentrations of chlorothalonil were highly efficient in management of the disease. The disease was completely controlled by chlorothalonil (> 250  µ g mL⁻¹), and in the presence of GSE 18 or GSE 19, 100  µ g mL⁻¹ chlorothalonil was equally effective. Application of rifamycin-resistant mutants of GSE 18 or GSE 19 together with chlorothalonil significantly increased the survival of these isolates in the groundnut phylloplane. In the field, a combination of GSE 18 and 500  µ g mL⁻¹ chlorothalonil reduced disease severity comparable to 2000  µ g mL⁻¹ chlorothalonil alone. Use of chlorothalonil-tolerant pseudomonads together with a quarter concentration of the recommended field dose of chlorothalonil doubled pod yield compared with the untreated unsprayed control.     

Improved fireblight diagnostics using quantitative real-time PCR detection of Erwinia amylovora chromosomal DNA

Specific and sensitive TaqMan real-time PCR assays were developed targeting chromosomal DNA of Erwinia amylovora ( ams C gene and ITS region). These assays increased the reliability of detection of E. amylovora strains, regardless of their plasmid profile, and have the ability to differentiate between Erwinia spp. strains from Hokkaido, Erwinia pyrifoliae and Erwinia spp. isolated from necrotic pear blossoms in Spain. The assays were used for testing the efficiency of three different extraction methods to remove plant-based PCR inhibitors. Combined with an automated DNA-extraction method based on magnetic beads (QuickPick™), the real-time PCR assays reliably detected at least 10³ cells mL^−l ( c. four cells per reaction) of the pathogen from blighted woody plant material. In testing of symptomless samples, absolute quantification of E. amylovora before and after enrichment in liquid media provided proof of E. amylovora viability and its ability to multiply, including in cases when subsequent isolation in pure culture was unsuccessful.     

Biological control of damping-off of sugar beet by Pseudomonas putida applied to seed pellets

Pseudomonas putida 40RNF applied to seed pellets reduced the occurrence of Pythium damping-off of sugar beet. A density of 6 × 10⁷ 40RNF per pellet reduced Pythium damping-off from 70 to 26% when seeds were sown in artificially infested soil (250 propagules Pythium ultimum per g dry soil). The efficacy of 40RNF was dependent on its density in the seed pellet (in the range 2 × 10⁴–6 × 10⁸ per pellet) and on the number of propagules of Pythium in soil. 40RNF declined to or stabilized at approximately 1 × 10⁶ per pellet 3 days after planting, and this was independent of the inoculum density. This indicated that the crucial steps resulting in damping-off of sugar beet caused by Pythium ultimum must occur within 3–4 days of sowing. 40RNF reduced pericarp colonization by P. ultimum by 43% 48 h after planting and caused a 68% decrease in the number of sporangia of P. ultimum in the surrounding soil (0.0–5.0 mm). P. putida 40RNF also reduced pre and post-emergence damping-off (from 69.5 to 37.5%) caused by indigenous populations of Pythium species in an infested soil and this was as effective as the fungicide hymexazol (69.5 to 40%).     

Antifungal activity of bergenin, a constituent of Flueggea microcarpa

The antifungal activity of bergenin against some plant pathogenic fungi, namely, Alternaria alternata , A. brassicae , A. carthami , Fusarium udum , F. oxysporum f.sp. ciceri , Curvularia lunata and Erysiphe pisi , was studied. Bergenin as its monosodium salt was effective against all the fungi and the effective dose for complete inhibition of spore germination varied from 15 μg mL⁻¹ for F. udum to 125 μg mL⁻¹ for E. pisi . Experiments on the effect of bergenin on powdery mildew development under glasshouse conditions revealed that it can control powdery mildew of pea at 2000 μg mL⁻¹ by postinoculation treatment, the results being comparable with those of carbendazim (1000 μg mL⁻¹) and wettable sulfur (2000 μg mL⁻¹). It affected hyphal elongation and the number of primary and secondary branches.     

Bioassays of glucosinolate-derived isothiocyanates against postharvest pear pathogens

The present study assayed the effect of six isothiocyanates (ITCs), produced by the enzymatic hydrolysis of glucosinolates, on fungal pathogens of pear. Sample pear fruits were artificially inoculated through induced wounds with conidial suspensions of Botrytis cinerea Rhizopus stolonifer Monilinia laxa Mucor piriformis or Penicillium expansum and were then treated with ITCs. Of the six ITCs tested, the ITC from glucoraphenin showed the highest effectiveness after 6 days at 20°C, against M. laxa B. cinerea and M. piriformis . The effectiveness of the ITC from glucoraphenin against M. laxa was assayed in two further trials to test the effect of ITC concentration on different concentrations of inoculum and to determine the duration of the curative effect of this ITC. ITC concentration directly affected fungus control capacity. The highest ITC concentration (3.6 mg mL⁻¹) afforded pathogen control at the highest level of pathogen concentration (10⁶ conidia mL⁻¹) after 6 days at 20°C. Its curative effect was evident up to 40 h after inoculation.     

Occurrence of Corynespora cassiicola isolates resistant to boscalid on cucumber in Ibaraki Prefecture, Japan

A total of 651 isolates of cucumber corynespora leaf spot fungus ( Corynespora cassiicola ) collected from cucumber in Japan, either with (438 isolates) or without (213 isolates) a prior history of boscalid use, were tested for their sensitivity to boscalid by using a mycelial growth inhibition method on YBA agar medium. Additionally, seven isolates of C. cassiicola obtained from tomato, soybean, eggplant (aubergine) and cowpea in different locations in Japan were tested before boscalid registration. Minimum inhibitory concentration (MIC) and 50% effective concentration (EC₅₀) values for 220 isolates from crops without a prior history of boscalid use ranged from 0·5 to 7·5 μg mL⁻¹ and from 0·04 to 0·59 μg mL⁻¹, respectively. Two hundred and fourteen out of 438 isolates collected from ten cucumber greenhouses in Ibaraki Prefecture, Japan, which received boscalid spray applications showed boscalid resistance, with MIC values higher than 30 μg mL⁻¹. Moreover, resistant isolates were divided into two groups: a moderately resistant (MR) group consisting of 189 isolates with EC₅₀ values ranging from 1·1 to 6·3 μg mL⁻¹, and a very highly resistant (VHR) group consisting of 25 isolates with EC₅₀ values higher than 24·8 μg mL⁻¹. MR isolates were detected from all ten greenhouses, but VHR isolates were detected from only three. As a result of fungus inoculation tests which used potted cucumber plants, control failures of boscalid were observed against resistant isolates. Efficacy of boscalid was remarkably low against VHR isolates in particular. This is the first known report on boscalid resistance in Japan.