Ascospore Germination Research Articles

Identification of Mycosphaerella species associated with Eucalyptus nitens leaf defoliation in South Africa

Eucalyptus nitens is an important plantation tree species in South Africa, where it is grown for paper and pulp production. The growth and performance of E. nitens in South Africa is, however, reduced substantially by Mycosphaerella leaf blotch (MLB) disease. The aim of this study was to determine which species of Mycosphaerella are associated with MLB epidemics on E. nitens in the KwaZulu-Natal Province of South Africa. Mycosphaerella species were isolated from leaves from two commercial E. nitens plantations by inducing the active release of ascospores onto agar. All isolates were identified according to their ascospore germination patterns, anamorph associations and sequence data generated from the Internal Transcribed Spacer region of the rDNA operon. From taxonomic and sequence data, M. nubilosa was identified as the main cause of MLB on E. nitens in commercial plantations in the KwaZulu-Natal Province. M. nubilosa is an important Eucalyptus leaf pathogen, and its correct identification in South Africa is important for the future planting of E. nitens. This is particularly true during the initial years of tree growth where MLB results in severe defoliation.

Dormant ascospores of Talaromyces macrosporus are activated to germinate after treatment with ultra high pressure.

Ascospores of Talaromyces macrosporus are constitutively dormant and germinate after a strong external shock, classically a heat treatment. This fungus is used as a model system to study heat resistance leading to food spoilage after pasteurization. This study evaluates the effect of high pressure on the germination behaviour of these spores. Ascospore containing bags were subjected to ultra high pressure and spores were plated out on agar surfaces. Untreated suspensions showed invariably very low germination. Increased germination of ascospores occurred after short treatments at very high pressure (between 400 and 800 MPa). Activation is partial compared with heat activation and did not exceed 6.9% (65 times that of untreated suspensions) of the spore population. Maximum activation was attained shortly (10 s-3 min) after the pressure was applied and accompanied by cell wall deformations as judged by scanning electron microscopy. The spores observed in this study were harvested from cultures that were 39-58 days old. The maturity of spores at similar developmental stages was measured by assessing the heat resistance of ascospores. Between 20 and 40 days heat resistance increased 2.4-fold, but only an additional increase of 1.3-fold was observed at later stages (40-67 days). Our investigations show that high pressure constitutes a second type of shock that can activate heat-resistant ascospores to germinate. Activation is maximal after very short treatments and accompanied with changes in the cell wall structure. High-pressure activation is not the result of immaturity of the ascospores. These observations are relevant for the application of high pressure as a novel pasteurization method.

Influência da temperatura e da luminosidade no desenvolvimento de Guignardia citricarpa, agente causal da mancha preta dos frutos cítricos

O presente trabalho teve como objetivo avaliar sob condições de laboratório, o efeito de temperatura (15, 18, 20, 21, 24, 25, 27, 30, 33 e 35 ± 1 ºC) nas condições escuro, luz contínua e fotoperíodo 12/12 h, na produção de pseudotécios de isolados de Guignardia citricarpa, provenientes de regiões cítricolas dos Estados de São Paulo e Rio de Janeiro. Discos de folhas de limoeiro 'Cravo' (Citrus limonia), de 12 mm de diâmetro, foram autoclavados e depositadas (parte abaxial) na superfície do meio de cultura constituído por ágar-água 2%. Foram colocados quatro discos de folhas por placa onde, de forma conjunta e intercalar aos mesmos, depositaram-se dois discos obtidos de colônias de Phyllosticta citricarpa, com 21 dias de incubação. Foi, também, estudado o efeito da temperatura e do tempo de incubação (2, 8 e 16 h) na germinação dos ascósporos. Após 21 dias de incubação, a ótima temperatura ajustada pela função beta generalizada, para produção de pseudotécios deu-se a 26 e 22,5 a 27,5 °C, sob condição de escuro e de luz, respectivamente. Observou-se também produção de pseudotécios a 27 ºC em fotoperíodo 12/12 h. Em estudo complementar foi verificado que, aos 19 dias, a 27 ºC, cerca de 90% dos pseudotécios haviam alcançado a maturidade, com abundante produção de ascósporos. A maior porcentagem de ascósporos germinados foi constatada na temperatura de 24 ºC, após 16 h de incubação. Dentre as vantagens alcançadas, incluem-se a possibilidade (i) da produção massal de ascósporos em curto período de tempo, e (ii) da padronização do inóculo, tanto qualitativa, quanto quantitativa.

Activation and Inactivation of Talaromyces macrosporus Ascospores by High Hydrostatic Pressure

The effect of high hydrostatic pressure treatment (with pressures of up to 700 MPa) on Talaromyces macrosporus ascospores was investigated. At 20°C, pressures of ≥200 MPa induced the activation and germination of dormant ascospores, as indicated by increased colony counts for ascospore suspensions after pressure treatment and the appearance of germination vesicles and tubes. Pressures of >400 MPa additionally sensitized the ascospores to subsequent heat treatment. At pressures of >500 MPa, activation occurred in a few minutes but was followed by inactivation with longer exposure. However, even with the most extreme pressure treatment, a fraction of the ascospore population appeared to resist both activation and inactivation, and the maximal achievable reduction of ascospores was on the order of 3.0 log10 units. Pressure-induced ascospore activation at 400 MPa was temperature dependent, with minimum activation at 30 to 50°C and ≥10-fold higher activation levels at 10 to 20°C and at 60°C, but it was not particularly pH dependent over a pH range of 3.0 to 6.0. Pressure inactivation at 600 MPa, in contrast, was pH dependent, with the inactivation level being 10-fold higher at pH 6.0 than at pH 3.0. Observation of pressure-treated and subsequently dried spores with the use of light and scanning electron microscopy revealed a collapse of the spore structure, indicating a loss of the spore wall barrier properties. Finally, pressure treatment sensitized T. macrosporus ascospores to cell wall lytic enzymes.

The Impact of Different Long‐Term Storage Conditions on the Viability of Lichen‐Forming Ascomycetes and their Green Algal Photobiont, Trebouxia spp.

Abstract: Lichen‐forming ascomycetes and their green algal photobionts completely die off within approximately 3 years of storage at room temperature. Macroscopically this is recognizable as a colour change, the green shades of the chlorophylls being lost. In fluorescent light microscopy preparations an increase in fungal autofluorescence and a significant decrease in chlorophyll autofluorescence in the Trebouxia cells was observed. In transmission electron microscopy preparations of Xanthoria parietina and its green algal photobiont, Trebouxia arboricola, the fungal membrane systems were found to be largely broken down whereas the shrivelled algal protoplast failed to rehydrate after storage at room temperature. When stored in the desiccated state at ‐ 20 °C, both partners of the symbiosis stayed fully viable for up to 13 years, their colouration and chlorophyll fluorescence being unchanged. Viability was measured as ascospore ejection and germination rates in Xanthoria parietina, soredium germination rates in Xanthoria fallax, Hypogymnia physodes and Parmelia sulcata, and autospore formation rate in Trebouxia cells (green algal photobiont), which had been isolated from the thalli after rehydration. Thallus fragments of Xanthoria parietina were shown to grow normally after one week of storage in LN2 without any cryoprotectant. In the desiccated state deep‐frozen samples can be repeatedly brought to room temperature and back to ‐ 20 °C without any loss of viability. Cryopreservation is therefore a suitable mode of long‐term storage of viable lichen thalli for experimental studies or transplant experiments.

Apothecia and Ascospores of Lobaria oregana and Lobaria pulmonaria Investigated

Apothecia of Lobaria oregana and L. pulmonaria emerge in late spring and discharge ascospores throughout the year. Most populations have a few fertile thalli, although the proportion of fertile thalli usually is less than 25 percent. Ascospores fail to germinate in water or on water agar but do germinate on agar containing an adsorbant and either a sugar or the sugar-alcohol ribitol. It is postulated that the ascospores of these species contain an autoinhibitor that must be removed before germination. Widespread ascospore germination in the presence of an adsorbant in Peltigera aphthosa, P. membranacea, and Pseudocyphellaria anthraspis, as well as in Lobaria, suggest that this phenomenon might be widespread in the Peltigerales.

Apothecia and ascospores of Lobaria oregana and Lobaria pulmonaria investigated

Apothecia of Lobaria oregana and L. pulmonaria emerge in late spring and discharge ascospores throughout the year. Most populations have a few fertile thalli, although the proportion of fertile thalli usually is less than 25 percent. Ascospores fail to germinate in water or on water agar but do germinate on agar containing an adsorbant and either a sugar or the sugar-alcohol ribitol. It is postulated that the ascospores of these species contain an autoinhibitor that must be removed before germination. Widespread ascospore germination in the presence of an adsorbant in Peltigera aphthosa, P. membranacea, and Pseudocyphellaria anthraspis, as well as in Lobaria, suggest that this phenomenon might be widespread in the Peltigerales.

Antagonism and biocontrol potential of Ulocladium atrum on Sclerotinia sclerotiorum

Antagonism and biocontrol potential of Ulocladium atrum against Sclerotinia sclerotiorum were investigated and compared to another biocontrol agent Coniothyrium minitans of S. sclerotiorum . Dual cultural assay on potato dextrose agar revealed that U. atrum was antagonistic to S. sclerotiorum . It could grow over the colony of S. sclerotiorum , but could not parasitize sclerotia of this pathogen. Results of spore germination tests on PDA and water agar (WA) showed that conidia of U. atrum or pycnidiospores of C. minitans had no inhibitory effects on the germination of ascospores of S. sclerotiorum. Conidia of U. atrum germinated more rapidly and germ tubes of U. atrum developed more extensively than ascospores of S. sclerotiorum and pycnidiospores of C. minitans . When petals of canola (cv Westar) were inoculated with spores of U. atrum or C. minitans at 5.4×10 4 spores/petal (at 24 h before, simultaneously, or 24 h after) and ascospores of S. sclerotiorum at 5.0×10 3 ascospores/petal and placed on detached leaves of canola, incidence of petal-mediated infection of leaves by S. sclerotiorum was significantly reduced. Moreover, the suppression of colonization of petals by S. sclerotiorum was related to the spore concentrations of these two biocontrol agents. Another test on alfalfa showed that application of U. atrum or C. minitans on senescent petals which remained attached to pods of alfalfa significantly reduced the infection of S. sclerotiorum on pods and seeds of alfalfa. This study suggests that the antagonist U. atrum is an effective biocontrol agent of S. sclerotiorum and the efficacy is similar to that of the mycoparasitic fungus C. minitans .

Effects of temperature on ascospore germination and penetration of oilseed rape (Brassica napus) leaves by A‐ or B‐groupLeptosphaeria maculans(phoma stem canker)

Ascospores of both A‐group and B‐groupLeptosphaeria maculansgerminated at temperatures from 5 to 20°C on leaves of oilseed rape. Germination of ascospores of both groups started 2 h after inoculation and percentage germination reached its maximum about 14 h after inoculation at all temperatures. Both the percentage of A‐/B‐group ascospores that had germinated after 24 h incubation and germ tube length increased with increasing temperature from 5 to 20°C. Germ tubes from B‐group ascospores were longer than those from A‐group ascospores at all temperatures, with the greatest difference at 20°C. Hyphae from ascospores of both groups penetrated the leaves predominantly through stomata, at temperatures from 5 to 20°C. A‐group ascospores produced highly branched hyphae that grew tortuously, whereas B‐group ascospores produced long, straight hyphae. The percentage of germinated ascospores that penetrated stomata increased with increasing temperature from 5 to 20°C and was greater for A‐group than for B‐groupL. maculansafter 40 h incubation.

Bgs3p, a Putative 1,3-β-Glucan Synthase Subunit, Is Required for Cell Wall Assembly in Schizosaccharomyces pombe

beta-Glucans are the main components of the fungal cell wall. Fission yeast possesses a family of beta-glucan synthase-related genes. We describe here the cloning and characterization of bgs3(+), a new member of this family. bgs3(+) was cloned as a suppressor of a mutant hypersensitive to Echinocandin and Calcofluor White, drugs that interfere with cell wall biosynthesis. Disruption of the gene is lethal, and a decrease in Bgs3p levels leads to rounded cells with thicker walls, slightly reduces the amount of the beta-glucan, and raises the amount of alpha-glucan polymer. These cells finally died. bgs3(+) is expressed in vegetative cells grown in different conditions and during mating and germination and is not enhanced by stress situations. Consistent with the observed expression pattern, Bgs3-green fluorescence protein (GFP-Bgs3p) was found at the growing tips during interphase and at the septum prior to cytokinesis, always localized to growth areas. We also found GFP-Bgs3p in mating projections, during the early stages of zygote formation, and at the growing pole during ascospore germination. We conclude that Bgs3p localization is restricted to growth areas and that Bgs3p is a glucan synthase homologue required for cell wall biosynthesis and cell elongation in the fission yeast life cycle.

Effect of soil temperature on disease development in melon plants infected by Monosporascus cannonballus

The effect of soil temperature on melon collapse induced by Monosporascus cannonballus was studied in the laboratory and in the field. In the laboratory, ascospore germination and hyphal penetration into melon roots were enhanced by increasing the temperature from 20 to 32°C. The optimum temperature for mycelial growth of five isolates of M. cannonballus was 30°C. In the field, the effect of temperature was studied in experiments conducted during the winter and autumn cropping seasons from 1995 to 1998. Disease progress was much faster in the autumn than in the winter crop seasons. Disease incidence reached 100% in the three consecutive autumn seasons studied. In the winter seasons, however, planting date influenced disease incidence. Early planting, at the beginning of January, resulted in a low disease incidence (6–26%, 125 days after planting), whereas planting at the end of January resulted in higher disease incidence (72–88%, 95–119 days after planting). In plots in which the soil was artificially heated to 35°C during the winter season, disease incidence reached 85%, as in the autumn season. Plants grown during the winter in unheated soil, or in artificially heated soil disinfected with methyl bromide, did not collapse. Root colonization by the pathogen was higher in the autumn and in heated soil than in the winter season in nonheated soil. Fifty per cent of root segments were colonized 35, 42 and 67 days after planting in the winter‐heated, autumn and winter‐unheated plots, respectively. A high correlation was found between soil temperatures above 20°C during the first 30 days after planting and disease severity. It is suggested that soil temperature during the early stages of plant development is an important factor in disease development and the expression of melon collapse caused by M. cannonballus.

Trehalose degradation and glucose efflux precede cell ejection during germination of heat-resistant ascospores of Talaromyces macrosporus.

Talaromyces macrosporus forms ascospores that survive pasteurization treatments. Ascospores were dense (1.3 g ml(-1)), relatively dry [0.6 g H(2)O (g dry weight)(-1)] and packed with trehalose (9-17% fresh weight). Trehalose was degraded to glucose monomers between 30 and 100 min after heat activation of the spores. The maximal activity of trehalase was calculated as 400-520 nmol glucose formed min(-1) (mg protein)(-1) as judged by measurements of the trehalose content of spores during germination. During early germination, glucose was released from the cell (10% of the cell weight or more). The intracellular concentration of glucose only peaked briefly. After 160-200 min, the protoplast encompassed by the inner cell wall was ejected through the outer cell wall in a very quick process. Subsequently, respiration of spores increased strongly. The data suggested that trehalose is primarily present for the protection of cell components as glucose is released from the cell. Then, an impenetrable outer cell wall is shed before metabolic activity increases.

The effect of temperature and wetness duration on infection and a warning system for European canker ( Nectria galligena) of apple in Chile

European canker ( Nectria galligena Bres.) is a major disease of apple in Chile, mainly producing cankers on twigs and branches. Disease incidence over 50% has been reported on ‘Richard Red’ apples in Southern Chile. In vitro, conidial germination of N. galligena was low at 6°C and 32°C, and optimum germination temperature was estimated between 20°C and 25°C. Ascospore germination was low at 5°C and rapidly increased as temperature increased from 5°C to 20°C. Germination rate, calculated as germination frequency over time at 20°C, was 2.3 times faster for ascospores than conidia, and germination of ascospores increased 2.6 times faster than conidia as temperature increased from 5°C to 20 °C. Infection caused by conidia of N. galligena through leaf scars was significantly dependent on temperature and free moisture duration. However, regardless of the wetness duration no infection was obtained at 5°C. This is in agreement with the lack of conidial germination obtained below 6°C by in vitro studies. Disease incidence increased linearly with temperatures between 5 and 20°C. Based on our results, a 2-h moisture period was enough to promote a significant infection at 20°C but longer moisture periods were needed at lower temperatures. An infection warning system, based on the analysis of weather parameters, was developed and implemented in the predictive software PatFrut ©. Over two seasons, the warning system was an effective tool for determination of the need of fungicide treatments against European canker. Significant differences in disease incidence and severity were obtained reducing disease incidence from over 24% in 1999 to 4.6% when treatments were schedule according to the model program. Differences in severity, but not disease incidence were found between the model program and the standard program. This forecast model assumed that conidia, produced during leaf fall were not a limiting factor. It also assumed that susceptible leaf scars were continuously available between March and July. The protective fungicide treatments applied during leaf fall significantly reduced infection in the next season suggesting that leaf scars are important infection courts for N. galligena on apples.

Sensitivity of Mycosphaerella fijiensis from Banana to Trifloxystrobin.

An ascospore germination method was developed and validated to assess the sensitivity of bulk samples of Mycosphaerella fijiensis to trifloxystrobin. Using this method, the sensitivity of 142 ascospore samples from banana plantations not treated with strobilurins was analyzed to establish a baseline of pathogen sensitivity. A bulk method was utilized for monitoring purposes because it avoids potential complications due to the isolation and propagation of single-spore isolates and enables the testing of larger samples. Following intensive use of strobilurins (6 to 11 applications per year) over 4 years, under conditions of high disease pressure and the absence of sanitary measures at a development site in Costa Rica, bulk samples with 50% effective concentration (EC50) resistance factors (RFs) in excess of 500 compared with the mean baseline sensitivity were detected. Single-ascospore isolates derived from spores germinating at the discriminatory dose of 3 μg/ml were also resistant, suggesting that the frequency of resistant individuals in bulk samples could be estimated from the relative numbers of ascospores growing at this dose. The resistance of selected isolates was confirmed in planta. In vitro tests with four resistant and two sensitive single-ascospore isolates collected from different locations and times indicated possible cross-resistance of trifloxystrobin to azoxystrobin, famoxadone, and fenamidone, but not to propiconazole.

Heteroallelism at the het-c Locus Contributes to Sexual Dysfunction in Outcrossed Strains of Neurospora tetrasperma

Neurospora tetrasperma is naturally heterokaryotic, with cells possessing haploid nuclei of both a and A mating types. As a result, isolates are self-fertile (pseudohomothallic). Occasional homokaryotic ascospores and conidia arise, however, and they produce strains that are self-sterile and must outcross to complete sexual reproduction. Invariably, laboratory crosses employing sibling a and A strains from the same parental heterokaryon restore the pseudohomothallic, heterokaryotic state. In contrast, outcrosses employing a and A strains from different wild isolates typically result in sexual dysfunction. Diverse sexual dysfunction types have been observed, ranging from complete sterility to reduced viability. We report that one type of dysfunction, characterized by spontaneous loss of the heterokaryotic state upon ascospore germination, can result from the interaction of incompatible alleles at heterokaryon incompatibility loci. Specifically, we demonstrate that homoallelism at the het-c locus in N. tetrasperma is required for heterokaryon stability. Heterokaryon incompatibility therefore provides an obstacle to outcrossing in this species, an observation with important implications for fungal life-cycle evolution.

The use of RAPD-PCR analysis for the differentiation of Mycosphaerella species from Eucalyptus in Australia

Mycosphaerella contains some of the most damaging foliar pathogens of Eucalyptus. Identification of the species has relied mainly on characters such as ascospore morphology and the mode of ascospore germination. Wide variation and overlap in morphological characters, especially ascospore shape and size, has lead to confusion in species concepts. This study examines the potential of RAPD-PCR to differentiate Mycosphaerella species that cause leaf diseases of Eucalyptus in Australia. RAPD-PCR was conducted on 39 isolates representing 10 species of Mycosphaerella collected from southern Australia. Primers were chosen mainly to distinguish between the two most common and damaging species in Australia, M. cryptica and M. nubilosa. The RAPD analysis revealed five groups within the 39 isolates, four of which represent distinct species: M. cryptica, M. gregaria, M. nubilosa and M. marksii. For these four species, the cluster analysis and RAPD patterns gave results that fitted well with classical taxonomic criteria. Variation within M. cryptica, M. nubilosa and M. marksii was low, even though the isolates within these species originated from diverse hosts and locations. The remaining species in this study grouped together and could not be easily separated using the RAPD data here.

Suppression of Sclerotinia sclerotiorum apothecial formation by the soil bacterium Serratia plymuthica: identification of a chlorinated macrolide as one of the causal agents

A selection of soil bacteria was screened for their ability to interfere with carpogenic germination of Sclerotinia sclerotiorum. Nine out of 300 bacterial isolates were found to significantly suppress apothecial formation. One of these isolates, identified as a strain of Serratia plymuthica, was highly effective in inducing complete suppression of apothecial formation at high concentrations, and also strongly inhibited the germination of ascospores as well as hyphal growth of S. sclerotiorum. A bioassay-guided purification procedure starting with the cell-free supernatant of the bacterial culture led to the identification of a chlorinated macrolide as an active compound able to induce the observed inhibition. Spectroscopic data showed the compound to be identical to haterumalide A. The data presented show the ability of this compound to inhibit apothecial formation and ascospore germination. Other possible mechanisms involved in inhibition of apothecial formation and mycelial and hyphal growth of S. sclerotiorum by the same isolate are discussed. The relevance of our observations to natural systems will be the subject of further research.

Detection of fungi in planta: effectiveness of surface sterilization methods

The effectiveness of surface sterilization methods were compared using root tissue of barley ( Hordeum vulgare ) colonized by two Chaetomium species in an aseptic plant growth system. The reliability of different sterilants to inhibit ascospore germination was tested. Ascospores on nitrocellulose membranes were either treated directly or ascospores adhering to axenic barley seedlings were treated on plant tissue. Inhibition of ascospore germination on nitrocellulose membranes was achieved with lower concentrations of sterilants than when the spores were on plant surfaces. A 10% peracetic acid treatment was necessary if experiments were conducted ad planta . The sterilants penetrated into epidermal root tissue and caused damage to this area, as shown by the vital dye DiOC 7 (3). It was concluded that effective surface sterilization techniques for ascospores adhering superficially to plant tissue were not appropriate for the detection of fungi growing within the epidermis.

ITS rDNA phylogeny of selected Mycosphaerella species and their anamorphs occurring on Myrtaceae

Species of Mycosphaerella and their anamorphs are commonly found on the leaves of Myrtaceae, many of which are defoliated by these pathogens. The taxonomy of these fungi has been based on minute morphological dierences, and virtually nothing is known regarding their relatedness to each other. In this study, we present a phylogeny of 30 species of Mycosphaerella or their anamorphs from myrtaceous hosts, based on sequence data from the ITS regions of the ribosomal RNA operon. Fifteen of the species were also analysed for the 5« end of the large subunit (28S), which produced a phylogeny similar to that obtained for the ITS data set. The Mycosphaerella species included in this analysis are all regarded as representatives of section Plaga, and appear to represent a monophyletic assemblage. Mycosphaerella lateralis was the only species shown to have a wide host range. In general, species clustered together based on their anamorph genera. Species with Colletogloeopsis and Stenella anamorphs always grouped in two respective clusters. However, species with Mycovellosiella, Phaeophleospora, Pseudocercospora and Uwebraunia anamorphs occurred separately, suggesting that they have evolved more than once within Mycosphaerella. Based on the ITS data set, all morphospecies were also shown to be phylogenetic species, although too few isolates were available to address questions relating to intraspecies variation. Nevertheless, ITS sequence data proved sucient to distinguish morphologically similar taxa that have hitherto only been distinguished based on ascospore germination patterns and anamorph characteristics. Sequence data presented in this study should facilitate the identification of Mycosphaerella species occurring on Myrtaceae in the future.

Fungal populations on sunflower (Helianthus annuus) anthosphere and their relation to susceptibility or tolerance to Sclerotinia sclerotiorum attack.

Analysis of the fungal flora from different floret parts of various sunflower (Helianthus annuus) varieties showed that there are differences in both fungal species and frequency, depending on whether the sunflower variety is susceptible (SV) or tolerant (TV) to attack of the flower heads by the ascomycete pathogen Sclerotinia sclerotiorum. The sunflower varieties analyzed were SV: HA 300 and Z 20028, and TV: HA 302, Z AV 8410 and Z 30629. The isolates showed different "in vitro" behavior as biocontrol agents. The most common types of interaction with Sclerotinia sclerotiorum were D2 and D2+ (hyphal contact) for isolates from SV and TV, while some of the isolates from TV displayed antibiosis. The microorganisms that colonize TV florets play a part in an indirect mechanism that protects flowers from ascospore germination and pathogen growth.