Fungicidal activity of essential oils of Cinnamomum zeylanicum (L.) and Syzygium aromaticum (L.) Merr et L.M. Perry against crown rot and anthracnose pathogens isolated from banana

AIMS: To develop a post-harvest treatment system against post-harvest fungal pathogens of banana using natural products. METHODS AND RESULTS: Colletotrichum musae was isolated and identified as the causative agent responsible for anthracnose peel blemishes while three fungi, namely Lasiodiplodia theobromae, C. musae and Fusarium proliferatum, were identified as causative agents responsible for crown rot. During the liquid bioassay, cinnamon [Cinnamomum zeylanicum (L.)] leaf, bark and clove [Syzygium aromaticum (L.)] oils were tested against the anthracnose and crown rot pathogens. The test oils were fungistatic and fungicidal against the test pathogens within a range of 0.03-0.11% (v/v). CONCLUSIONS: Cinnamon and clove essential oils could be used as antifungal agents to manage post harvest fungal diseases of banana. SIGNIFICANCE AND IMPACT OF THE STUDY: Cinnamon and clove essential oil could be used as alternative post-harvest treatments on banana. Banana treated with essential oil is chemically safe and acceptable to consumers. Benomyl (Benlate), which is currently used to manage fungal pathogens, can cause adverse health effects and could be replaced with volatile essential oils.     

Susceptibility of the bruchid Callosobruchus maculatus (Coleoptera: Bruchidae) and its parasitoid Dinarmus basalis (Hymenoptera: Pteromalidae) to three essential oils

The bruchid Callosobruchus maculatus (F.) causes major losses during the storage of seeds of Vigna unguiculata (Walp.) in West Africa. An endemic parasitoid, the pteromalid Dinarmus basalis (Rond.) reduces the increase in bruchid populations in stores and could be used for biological control. African farmers often introduce essential oils into granaries at harvest time. In Togo, essential oils were extracted from two Gramineae, Cymbopogon nardus (L.) and Cymbopogon schoenanthus (L.) and from a Lamiaceae, Ocimum basilicum (L.). The major components of these essential oils were citronellal in C. nardus, carene-2 and piperitone in C. schoenanthus and estragol in O. basilicum. Cymbopogon schoenanthus was the most toxic oil for C. maculatus adults. D. basalis adults were more susceptible to the three essential oils than the adults of their hosts C. maculatus. In the presence of cowpea seeds, the LC50s of the three essential oils were lower than in their absence, suggesting that the seeds may absorb a part of the volatiles. High doses of three essential oils slightly affected the survival of the fourth instar or the pupae of C. maculatus. This high survival was due to protection of larvae from volatiles by the surrounding seeds. The D. basalis were more affected by the oil volatiles than their hosts. Sub-lethal doses of essential oils reduced the duration of the adult life of both insect species and fecundity of the females. The differences in sensitivity of the host and its parasitoid could influence their population dynamics. The introduction of the essential oils into storage systems potentially could reduce density of parasitoid populations and increase seed losses.     

香茅精油对桔小实蝇产卵驱避作用及其化学成份分析

研究了香茅Cymbopogon nardus精油对桔小实蝇Bactrocera dorsalis (Hendel)的产卵驱避作用及其化学成分。结果表明,香茅精油对桔小实蝇产卵具有一定的驱避作用,不同浓度香茅精油处理芒果上的产卵量均显著低于对照,且随着香茅精油浓度的提高其产卵量逐渐减少,当浓度为10000μg/mL时,产卵驱避率为70.06%。采用气相色谱—质谱联用(GC-MS)技术分析了香茅精油的化学成份,结果发现该精油挥发物中含量较高的是3-蒈烯,香茅醛,香叶醇,香茅醇,柠檬烯。     

Essential Oils as Potent Source of Nematicidal Compounds

Bioassay tests were conducted to find out the nematicidal activity of eight essential oils against Meloidogyne incognita (Kofoid and White) Chitwood at four concentrations. Maximum activity was recorded in oils of Eucalyptus citriodora , Eucalyptus hybrida and Ocimum basilicum followed by Pelargonium graveolens , Cymbopogon martinii, Mentha arvensis, Mentha piperita and Mentha spicata oils, respectively. The eucalyptus ( E. citriodora and E. hybrida ) and Indian basil ( O. basilicum ) oils were highly toxic to M. incognita even at the lower concentrations, namely 500 and 250 ppm. The remaining oils were also toxic to the nematode but at different amounts.     

甘肃大葱贮藏期镰孢菌腐烂病病原鉴定

【目的】大葱在贮藏期频繁发生镰孢菌腐烂病,损失严重。明确该病害病原种类对病害防治具有重要意义。【方法】利用组织分离法对采集自甘肃省兰州市(区)蔬菜市场的16份大葱贮藏期镰孢菌腐烂病病样进行病原物的分离、纯化培养,经单孢分离后根据形态学特征,再结合r DNA-ITS、EF-1a(tef)基因序列分析的方法进行鉴定。【结果】共分离得到80株镰孢菌,经鉴定分属3个种,即层出镰孢菌(Fusarium proliferatum)、尖孢镰孢菌(F.oxysporum)和燕麦镰孢菌(F.avenaceum),其中层出镰孢菌为大葱镰孢菌腐烂病的优势致病菌,分离频率为52.50%。对兰州白葱不同部位进行致病性测定,结果表明层出镰孢菌对大葱鳞茎的致病力最强,而燕麦镰孢菌对大葱鳞茎的致病力最弱。【结论】3种镰孢菌作为该病害的病原,属国内首次报道。     

Fungitoxic activity of some essential oils

Of the five essential oils screened against Helminthosporium oryzae, the oils of Cymbopogon martinii (ginger grass oil), Cymbopogon oliveri, Cymbopogon sp. (rosa sofia oil) and Trachyspermum ammi (dethymolysed oil) exhibited strong fungitoxicity and showed wide range of activity. The oils were found more active than some of the prevalent synthetic fungicides and thus might be exploited as natural fungicides if successful infield trials. Besides, these oils were found toxic to various human pathogens.     

Catecholamine biosynthesis pathway potentially involved in banana defense mechanisms to crown rot disease

Variations in Cavendish bananas susceptibility to crown rot disease have been observed (Lassois et al., 2010a), but the molecular mechanisms underlying these quantitative host-pathogen relationships were still unknown. The present study was designed to compare gene expression between bananas (Musa acuminata, AAA, 'Grande-Naine') showing a high post-harvest susceptibility (S+) and bananas showing a low post-harvest susceptibility (S-) to crown rot disease. This comparison was performed between crowns (S+ and S-) collected one hour before standardized artificial inoculations with Colletotrichum musae. Fruit susceptibility was evaluated through lesion size on the crown 13 days later. Gene expression comparisons were performed with the cDNA-AFLP technique (Lassois et al., 2009). This revealed that a gene showing a strong homology with a dopamine-beta-monooxygenase (DoH) is differently expressed between S+ and S (Lassois et al., 2011). Furthermore, semi-quantitative real-time RT-PCR analyses between S+ and S- were applied to confirm the differential expression results for DoH obtained by cDNA-AFLP. Two biological replicates were tested. These semi-quantitative analyses were performed not only on tissues collected one hour before C. musae inoculation but also on crown tissues collected 13 days after inoculation. The real-time RT-PCR confirmed that DoH was upregulated in the S tissues collected at harvest, just before C. musae inoculation. This gene was also highly upregulated in the S- tissues collected 13 days after crown inoculation. Similar results were obtained for both biological replicates. Our results suggest that catecholamine's could play a role in banana defense mechanisms to crown rot disease.     

Specific detection of the toxigenic species Fusarium proliferatum and F. oxysporum from asparagus plants using primers based on calmodulin gene sequences

Fusarium proliferatum and Fusarium oxysporum are the causal agents of a destructive disease of asparagus called Fusarium crown and root rot. F. proliferatum from asparagus produces fumonisin B1 and B2, which have been detected as natural contaminants in infected asparagus plants. Polymerase chain reaction (PCR) assays were developed for the rapid identification of F. proliferatum and F. oxysporum in asparagus plants. The primer pairs are based on calmodulin gene sequences. The PCR products from F. proliferatum and F. oxysporum were 526 and 534 bp long, respectively. The assays were successfully applied to identify both species from the vegetative part of the plants.     

Effects of Cymbopogon nardus (L.) W. Watson essential oil on the growth and morphogenesis of Aspergillus niger

The growth inhibitory effect of Cymbopogon nardus (L.) W. Watson var. nurdus essential oil on Aspergillus niger (Van Tieghem) mycelium was determined on agar medium. The mycelium growth was completely inhibited at 800 mg/L. This concentration was found to be lethal under the test conditions. Essential oil at 400 mg/L caused growth inhibition of 80% after 4 days of incubation, and a delay in conidiation of 4 days compared with the control. Microscopic observations were carried out to determine the ultrastructural modifications of A. niger hyphae after treatment with C. nardus essential oil. The main change observed by transmission electron microscopy concerned the hyphal diameter and the hyphal wall, which appeared markedly thinner. These modifications in cytological structure might be caused by the interference of the essential oil with the enzymes responsible for wall synthesis which disturb normal growth. Moreover, the essential oil caused plasma membrane disruption and mitochondrial structure disorganization. The findings thus indicate the possibility of exploiting Cymbopogon nardus essential oil as an effective inhibitor of biodegrading and storage-contaminating fungi.     

Genetic Variability of Phytopathogenic Fusarium proliferatum Associated with Crown Rot in Asparagus officinalis

Fusarium proliferatum (teleomorph: Gibberella intermedia ) is a causal agent of crown rot of Asparagus officinalis and is one potential fumonisin-producing species within the genus Fusarium . It colonizes roots and crowns of asparagus plants, but could also be isolated from symptomless asparagus spears. Fusarium proliferatum isolates obtained from perennial asparagus plantings from Austria and Germany were included in a study on detectability and variability of two essential genes of the fumonisin-gene cluster. Genetic fingerprinting of 45 isolates revealed 14 different fingerprint groups, indicating genetic heterogenicity of F. proliferatum . Most isolates differentiated into three main fingerprint clusters, but no association was found between fingerprint group and origin of the isolates. By gene-specific PCR it was shown that, in 25 isolates tested, both initial genes of the fumonisin biosynthetic pathway – FUM1 , encoding a polyketide synthase and FUM8 , a gene for a putative aminoacyl transferase – were detectable. This suggests that these isolates were able to produce fumonisins and could contribute to the detected contamination in originating asparagus spears with this mycotoxin. Thus, early detection of FUM -genes in F. proliferatum -colonized asparagus may be suited to prevent uptake of fumonisin contaminated food with the human diet. Restriction fragment length polymorphism analysis (PCR-RFLP) of the amplified FUM gene fragments revealed little sequence variability, suggesting a conserved structure of these genes within this species. However, sequence analysis confirmed intraspecific nucleotide polymorphisms of these genes.     

Antifungal activity of essential oils from Cymbopogon: inter- and intraspecific differences

The influence of the genetic background of Cymbopogon species on the antifungal activity of essential oils derived from the plants was investigated against three yeast-like and nine filamentous fungi. Essential oils from distinct strains of the aromatic grass Cymbopogon showed interspecific and intraspecific differences in antifungal activity.     

香蕉上的镰孢菌种类及其系统发育关系(英文)

镰孢菌属真菌是香蕉上的重要病原菌,主要引起香蕉枯萎病以及香蕉冠腐病,在我国已明确引起香蕉枯萎病的病原为尖孢镰孢古巴专化型 Fusarium oxysporum f. sp. cubense(FOC)1号和4号生理小种,但是引起香蕉冠腐病的镰孢菌种类还未明确。为了解香蕉上镰孢菌在种间及种内水平上的多样性,2008–2011 年间作者从华南地区不同的水果市场及香蕉果园采集香蕉样品90份,分离得到143株镰孢菌。通过形态学观察及基于 EF-1α基因的系统进化分析鉴定出10种镰孢菌,即F. oxysporum、F. solani、F. camptoceras、F. pallidoroseum、F. stiloides、F. chlamydosporum、F.verticillioides、F. proliferatum、F. concentricum、F. sacchari,以及藤仓赤霉复合种(Gibberella fujikuroi species complex,GFC)中 3 个未定名的类群。轮纹镰孢 F. concentricum 及甘蔗镰孢 F.sacchari 是香蕉果实中最常见种,前菌为我国首次报道,后菌是首次报道与香蕉有关。对从香蕉上分离的藤仓赤霉复合种(GFC)及尖孢镰孢复合种(FOSC)的EF-1α序列进行了系统发育分析,其GFC中的27个菌株组成的单系群可分为7个不同的亚群,分别为 F.verticillioides、F. proliferatum、F. concentricum、F. sacchari 以及3个没有描述过的菌系 Fusarium sp. 1、Fusarium sp.2和 Fusarium sp.3;FOSC中的50个菌株形成2大分枝共12个谱系,分离自我国华南地区的21株尖孢镰孢形成7个谱系,其中 13株已知的香蕉枯萎病病原菌分布在3个谱系中,我国大陆的香蕉枯萎病病原菌菌株与来源于台湾地区及东南亚的菌株亲缘关系较近,FOC1号生理小种的遗传分化大于4号生理小种,FOC 1号生理小种与分离自香蕉果实上的尖孢镰孢菌的亲缘关系比与FOC 4号生理小种的亲缘关系更近。研究结果表明,我国香蕉上存在着丰富的镰孢菌种类,而且种内遗传多样性丰富。     

桂产罗勒挥发油化学成分的分析

采用GC-MS法建立桂产罗勒挥发油的化学成分分析方法。采用水蒸汽蒸馏法提取罗勒挥发油,用GC-MS联用技术分离并鉴定其成分,并用面积归一化法测定成分的相对含量。共鉴定出54个化合物,占挥发油总量的90.21%。桂产罗勒挥发油的主要成分是对烯丙基茴香醚、(+)表-双环倍半水芹烯、3,7,11-三甲基-(Z,E)-1,3,6,10-十二碳四烯,与文献报道的其他产地的罗勒油的化学成分存在显著差异。     

Comparative screening of plant essential oils: phenylpropanoid moiety as basic core for antiplatelet activity

Essential oils extracted from different plants (Anthemis nobilis L., Artemisia dracunculus L., Cannabis sativa L., Cupressus sempervirens L., Cymbopogon citratus (DC.) Stapf., Curcuma longa L., Foeniculum vulgare L., Hypericum perforatum L., Hyssopus officinalis L., Mentha spicata L., Monarda didyma L., Ocimum basilicum L., Ocotea quixos Kosterm., Origanum vulgare L., Pinus nigra J.F. Arnold, Pinus silvestris L., Piper crassinervium Kunth., Rosmarinus officinalis L., Salvia officinalis L., Salvia sclarea L., Santolina chamaecyparissus L., Thymus vulgaris L., Zingiber officinaie L.) were screened in guinea pig and rat plasma in order to assess antiplatelet activity and inhibition of clot retraction. The oils were chemically analysed and a relationship between components and ability to affect hemostasis was evidenced. O. quixos, F. vulgaris, and A. dracunculus showed the highest antiplatelet activity against ADP, Arachidonic Acid and the Thromboxane A2 agonist U46619 (IC50, 4-132 microg ml(-1)), and a good ability to destabilize clot retraction (IC50, 19-180 microg ml(-1)). For these oils a significant correlation between antiplatelet potency and phenylpropanoids content (54-86%) was evidenced thus suggesting a key role for this moiety in the prevention of clot formation. These findings provide the rationale to take in account the antiplatelet activity in the pharmacological screening of natural products containing phenylpropanoids.     

Comparison of bacteriostatic and bactericidal activity of 13 essential oils against strains with varying sensitivity to antibiotics

Aims:  To compare the bacteriostatic and bactericidal activity of 13 chemotyped essential oils (EO) on 65 bacteria with varying sensitivity to antibiotics.
Methods and Results:  Fifty-five bacterial strains were tested with two methods used for evaluation of antimicrobial activity (CLSI recommendations): the agar dilution method and the time-killing curve method. EO containing aldehydes ( Cinnamomum verum bark and Cymbopogon citratus ), phenols ( Origanum compactum , Trachyspermum ammi , Thymus satureioides , Eugenia caryophyllus and Cinnamomum verum leaf) showed the highest antimicrobial activity with minimum inhibitory concentration (MIC) <2% (v/v) against all strains except Pseudomonas aeruginosa . Alcohol-based EO ( Melaleuca alternifolia , Cymbopogon martinii and Lavandula angustifolia ) exhibited varying degrees of activity depending on Gram status. EO containing 1·8-cineole and hydrocarbons ( Eucalyptus globulus , Melaleuca cajeputii and Citrus sinensis ) had MIC_90% ≥ 10% (v/v). Against P. aeruginosa , only C. verum bark and O. compactum presented MIC ≤2% (v/v). Cinnamomum verum bark, O. compactum , T. satureioides , C. verum leaf and M. alternifolia were bactericidal against Staphylococcus aureus and Escherichia coli at concentrations ranging from to 0·31% to 10% (v/v) after 1 h of contact. Cinnamomum verum bark and O. compactum were bactericidal against P. aeruginosa within 5 min at concentrations <2% (v/v).
Conclusions:  Cinnamomum verum bark had the highest antimicrobial activity, particularly against resistant strains.
Significance and Impact of the Study:  Bacteriostatic and bactericidal activity of EO on nosocomial antibiotic-resistant strains.     

Induction of systemic resistance by mixtures of antagonist bacteria for the management of crown rot complex on banana

Among nine native bacterial strains isolated from banana fruit surface and rhizosphere and six bacterial strains introduced from the culture collection, three native strains viz., non-fluorescent Pseudomonas (NFP₆), Pseudomonas fluorescens (Pf3a), and Bacillus subtilis (BS₁); and two bacterial strains from culture collection viz., Azospirillum (AS1) and Azotobacter (AZ1) have recorded maximum inhibition of mycelial growth of crown rot pathogens (Lasiodiplodia theobromae and Colletotrichum musae) under in vitro condition. When these effective bacterial strains were treated on banana fruits under in vivo, significant reduction of crown rot disease and increased shelf life of banana was observed. However, bacterial strains applied as three way combinations (NFP₆ + Pf3a + BS₁) had greater effect compared with individual and two way combination of bacterial antagonist treatments. The effect of crown rot disease reduction was also comparable to that of fungicide Benomyl (0.1%) both under cold and room temperature storage conditions. Besides, the induction of defense-related enzymes such as phenylalanine ammonia-lyase (PAL), peroxidase (PO), polyphenoloxidase (PPO), and the accumulation of phenolics in banana fruit due to the application of bacterial antagonists were also studied at five different time intervals viz. 0th, 1st, 3rd, 5th and 7th days after treatment. When banana fruits treated with bacterial antagonists (individually and also in different combinations) and challenge-inoculated with crown rot pathogens, up to fourfold increase in defense-related enzymes and 3.6 fold increase in phenolic content was observed compared with control. The activity of these defense-related enzymes and phenolic content had gradually increased from 1st day after treatment to 3rd after treatment and reached their peak on 5th day after treatment. Among the bacterial antagonists which have been applied individually and in different combinations, the banana fruits treated with three-way antagonist mixture, i.e., NFP₆ + Pf3a + BS₁ recorded maximum induction of defense-related enzymes and accumulation of phenolics compared with individual and two-way combination of antagonist mixtures. This study suggest that the increased induction of defense-related enzymes and phenolic content due to the treatment of banana fruits with bacterial antagonists might have involved in the reduction of crown rot severity and in turn increased the shelf life of banana fruits.     

Five essential oils from aromatic plants of Cameroon: their antibacterial activity and ability to permeabilize the cytoplasmic membrane of Listeria innocua examined by flow cytometry

AIMS: To investigate the antibacterial effect of five essential oils (EO) extracted from aromatic plants (Cymbopogon citratus, Ocimumbasilicum, Ocimum gratissimum, Thymus vulgaris and Zingiber officinale) of Cameroon against strains of Listeria monocytogenes, L. innocua and Staphylococcus aureus. The ability of selected EO to permeabilize the cytoplasmic membrane of L. innocua was also examined. METHODS AND RESULTS: The antibacterial activity of the EO determined by the agar diffusion method showed that T. vulgaris had the highest activity followed by O. gratissimum and C. citratus. Lowest activity was recorded from Z. officinale and O. basilicum. Significant differences in sensitivity between strains of Listeria and S. aureus were observed. Flow cytometry of L. innocua stained with carboxy-fluorescein diacetate showed that the fluorescence intensity of cells exposed to EO decreased faster than nonexposed cells, indicating that EO permeabilized the cytoplasmic membrane with the leakage of carboxy-fluorescein. CONCLUSIONS: Almost all the EO tested showed antibacterial activity to a different extent. The antibacterial effect was due to permeabilization of the cytoplasmic membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has identified the preservative potential of the EO examined. The use of sensitive method, such as flow cytometry, is advantageous for quick generation of data on the antibacterial effect of EO.     

Nematicidal activity of essential oils and volatiles derived from Portuguese aromatic flora against the pinewood nematode, Bursaphelenchus xylophilus

Twenty seven essential oils, isolated from plants representing 11 families of Portuguese flora, were screened for their nematicidal activity against the pinewood nematode (PWN), Bursaphelenchus xylophilus. The essential oils were isolated by hydrodistillation and the volatiles by distillation-extraction, and both were analysed by GC and GC-MS. High nematicidal activity was achieved with essential oils from Chamaespartium tridentatum, Origanum vulgare, Satureja montana, Thymbra capitata, and Thymus caespititius. All of these essential oils had an estimated minimum inhibitory concentration ranging between 0.097 and 0.374 mg/ml and a lethal concentration necessary to kill 100% of the population (LC(100)) between 0.858 and 1.984 mg/ml. Good nematicidal activity was also obtained with the essential oil from Cymbopogon citratus. The dominant components of the effective oils were 1-octen-3-ol (9%), n-nonanal, and linalool (both 7%) in C. tridentatum, geranial (43%), neral (29%), and β-myrcene (25%) in C. citratus, carvacrol (36% and 39%), γ-terpinene (24% and 40%), and p-cymene (14% and 7%) in O. vulgare and S. montana, respectively, and carvacrol (75% and 65%, respectively) in T. capitata and T. caespititius. The other essential oils obtained from Portuguese flora yielded weak or no activity. Five essential oils with nematicidal activity against PWN are reported for the first time.     

Residual and ovicidal efficacy of essential oil‐based formulations in vitro against the donkey chewing louse Bovicola ocellatus

Essential oils have shown good experimental potential as novel veterinary ectoparasiticides. However, if they are to be used as veterinary products, they must be available in formulations that are suitable for practical application against specific ectoparasites. Here, the efficacies of formulations containing 5% (v/v) lavender or tea tree oil, in combination with two emulsifiers [a surfactant, 5% (w/v) N‐lauroylsarcosine sodium salt (SLS), and a soluble polymer, 5% (w/v) polyvinylpyrrolidone (PVP)], with or without 10% coconut oil, were tested in contact bioassays against the donkey chewing louse Bovicola ocellatus (Piaget) (Phthiraptera: Trichodectidae). Residual activity was quantified in open and closed containers; ovicidal efficacy was also examined. Exposure to either of 5% (v/v) lavender or tea tree oils with SLS or PVP resulted in louse mortality of 100%, but when coconut oil was included as an excipient, significantly lower efficacy was recorded. However, the formulations became significantly less effective after 2 h in open containers and 40 h in closed containers. The results confirm that the residual activity of essential oils is relatively transitory and the addition of 10% coconut oil does not prolong the period of insecticidal activity by slowing essential oil evaporation. Too short a period of residual activity is likely to be a significant impediment to the effective practical use of essential oils. However, unlike many synthetic pediculicides, the essential oils tested here were highly ovicidal, which suggests that prolonged residual activity may not be essential to kill newly hatched nymphs after treatment.     

Sources of crown rot (Phytophthora cactorum) infection in strawberry and the effect of cold storage on susceptibility to the disease

Cold-stored plants of strawberry cultivars Tamella, Cambridge Favourite and Redgauntlet were more susceptible to pathogenic isolates of Phytophthora cactorum than similar plants which had not been cold-stored. Indigenous nonpathogenic isolates of P. cactorum did not cause crown rot in cold-stored plants, although a small number of symptomless latent infections occurred. The majority of P. cactorum isolates causing crown rot symptoms were taken from infected strawberry crowns, although two isolates from gooseberry plants, but of uncertain origin, were also pathogenic. Outbreaks of crown rot in areas with no previous history of the disease therefore probably result from the importation of non-indigenous inoculum with planting material. Assessments of the timing of infection in relation to cold storage revealed that a high incidence of death in the cold store and chronic wilt symptoms on planting from the store resulted from initiating symptomless infections prior to cold storage. However, infection during the period immediately after cold storage resulted in rapid wilt symptoms of Phytophthora crown rot. When plated in sterile distilled water for 24 h, pieces of tissue from infected plants which had died during cold storage produced large numbers of sporangia and zoospores. This indicates that such plant material could provide a potent source of inoculum for infections in the post storage thawing environment. It is proposed that a combination of heightened host susceptibility resulting from cold storage and the presence of scatted latent infections or infected debris among the plants could result in a sudden, large scale appearance of crown rot, as sometimes is seen with cold-stored plantings of strawberries.