Worldwide Phylogeography of the Citrus Brown Spot Pathogen, Alternaria alternata

ABSTRACT Sixty-five isolates of Alternaria alternata were sampled from brown spot lesions on tangerines and mandarins (Citrus reticulata) and tangerine x grapefruit (C. reticulata x C. paradisi) hybrids in the United States, Colombia, Australia, Turkey, South Africa, and Israel to investigate the worldwide phylogeography of the fungus. Genetic variation was scored at 15 putative random amplified polymorphic DNA (RAPD) loci and 465 bp of an endo-polygalacturonase (endo-PG) gene was sequenced for each isolate. Cluster analysis of RAPD genotypes revealed significant differentiation between United State and Colombia isolates and Turkey, South Africa, Israel, and Australia isolates. Sequencing of endo-PG revealed 21 variable sites when the outgroup A. gaisen (AK-toxin-producing pathogen of Japanese pear) was included and 13 variable sites among the sampled isolates. Nucleotide substitutions at 10 of 13 variable sites represented silent mutations when endo-PG was translated in frame. Eight distinct endo-PG haplotypes were found among the sampled isolates and estimation of a phylogeny with endo-PG sequence data revealed three clades, each with strong bootstrap support. The most basal clade (clade 1) was inferred based on its similarity to the outgroup A. gaisen and consisted exclusively of pathogenic isolates from the United States and Colombia. Clade 2 consisted of pathogenic and nonpathogenic isolates from the United States, Australia, South Africa, and Israel and clade 3 contained pathogenic and nonpathogenic isolates from Australia, South Africa, Israel, and Turkey. Quantitative estimates of virulence (disease incidence) were obtained for isolates from the United States, Colombia, South Africa, Israel, and Turkey by spray inoculating detached citrus leaves and counting the number of lesions 24 h after inoculation. Large differences in virulence were detected among isolates within each location and isolates from the United States were significantly more virulent than isolates from other locations. Several isolates from Colombia, South Africa, Israel, and Turkey had low virulence and 8% of all isolates were nonpathogenic. All but one of the nonpathogenic isolates were found in clade 2 of the endo-PG phylogeny, which also included the most highly virulent isolates sampled.     

Environmental Factors Affecting Pseudothecial Development and Ascospore Production of Mycosphaerella citri, the Cause of Citrus Greasy Spot

ABSTRACT Mycosphaerella citri, the cause of citrus greasy spot, produces pseudothecia and ascospores in decomposing leaf litter on the grove floor. In laboratory studies, the effect of wetting and drying and temperature on the formation, maturation, and production of pseudothecia and ascospores was evaluated on mature, detached grapefruit leaves. Production of pseudothecia was most rapid when leaves were soaked five times per week for 2 h per day, but pseudothecial density and total ascospore production were greatest when leaves were soaked three times per week for 2 h per day. In duration of wetting studies, 3 h per day, 3 days per week brought about the most rapid production, but 10 to 30 min per day resulted in production of the most pseudothecia and ascospores. Pseudothecia and ascospore production were greatest at 28 degrees C and declined rapidly at lower and higher temperatures. Maturation of pseudothecia was slow at 20 and 24 degrees C, but production was high at 24 degrees C; at 32 degrees C, pseudothecia matured rapidly, but degenerated quickly. No mature pseudothecia were produced on leaves maintained continuously under wet conditions. In field studies, leaves were placed on the grove floor monthly from April 2000 to September 2001. Pseudothecia production was rapid during the summer rainy season from June to September. Pseudothecia produced on leaves placed in the grove from October to May developed and matured more slowly but were produced in much larger numbers than in summer. The number of days to first pseudothecial initials, 50% maturation, first discharge of ascospores, leaf decomposition, as well as pseudothecial density and incidence, were negatively related to average temperature. Total ascospore production was unrelated to temperature.     

影响链格孢菌生长及产孢的因子

在实验室研究了影响稗草病原真菌链格孢生长及产孢的因子.链格孢菌可在较宽的温度范围内生长、繁殖;连续黑暗、空气充足条件下有利于产孢;该菌在4种不同的植物产品上培养可产生大量的孢子,最多的无芒稗籽上每克干物质产孢量达3.59×108个,这些培养物在首次产孢收获后均可直接培养作二次产孢利用.固体(菌块)和液体(菌悬液)接种培养物均能成功产孢.但培养量大时液体接种优于固体接种,产孢量与接种菌悬液浓度呈正相关.     

Alternaria Brown Spot of Minneola in Greece; Evaluation of Citrus Species Susceptibility

During the last three years, a new disease was observed in northwestern Greece on Minneola trees, hybrid of mandarin and grapefruit. On May small brown necrotic leaf spots surrounded by yellow halo areas of various sizes appeared and covered a major portion of the leaves with extension of necrosis into the veins. On young fruits small, slightly depressed black spots were the first symptoms, which later became 2–7 mm in diameter. Brown spots were observed on the leaves and fruits in several orchards in the same area, causing leaves and fruits to drop. In some orchards over 50% of the fruits were affected. From the fruit and leaf spots the typical small-spore species Alternaria alternata was isolated. Pathogenicity tests were performed by artificially inoculating fruits of Minneola, common mandarin and Clementine. The symptoms of the disease were reproduced only on fruits of Minneola hybrids by the specific strain of the fungus Alternaria alternata pv. citri. Different citrus susceptibility tests indicated that mandarins Minneola, Nova and Page were very susceptible to tested isolates while Clementine SRA and Poros Clementine were not. All lemons and lime Seedless were not susceptible. Grapefruit New Hall was not susceptible, while the Star Ruby was. Orange Lane Late, Navel Late, Oval Poros, Olinda, Navel Athos were not susceptible and only Moro showed reaction being slightly susceptible only to one isolate.     

木霉几丁质酶对烟草赤星病菌的作用

以指形管培养法分别测定几丁质酶粗酶液和纯化的几丁质酶混合液(2种几丁质酶)对烟草赤星病菌孢子萌发的抑制作用。结果表明,较高浓度(25 2U)几丁质酶粗酶液在48h内强烈抑制孢子萌发和芽管伸长,或致芽管畸形和细胞壁破裂;几丁质酶混合液对赤星病菌的孢子萌发也表现出明显的抑制作用,但在相同或相近酶活性条件下,纯化的几丁质酶混合液(9 4U)和粗酶液(12 6U)对赤星病菌孢子萌发的抑制率(处理24h时)分别为46%和84 3%,前者明显低于后者。采用孢子液悬滴法接种烟苗(K 326)叶片测定木霉几丁质酶对赤星病菌致病性的影响。结果表明,几丁质酶粗酶液浓度越高,对孢子萌发抑制时间越长,抑制率越高;其浓度为4 9、9 8、19 5U/ml的抑制率7d时分别为36 8%、56 2%和57 6%。     

烟草赤星病拮抗菌株B75产生抗菌物质的条件

从烟草根际分离筛选到一株可产生抗烟草赤星病菌物质的枯草芽孢杆菌菌株B75。在以NB培养液进行试验时发现，B75在对数生长的中期开始产生抗菌物质，在生长的衰退期前存在一个产生抗菌物质的高峰期；在25～50℃、pH4．0～8．5范围内均可产生抗菌物质，其中30℃和pH6．0的培养条件下最适。不同的装液量试验结果表明，在三角瓶装液量为100ml／500ml时，是B75菌株菌体生长及产生抗菌物质的最适宜量。不同的碳、氮源试验结果表明，甘露醇、酵母膏最有利于B75菌株菌体生长，且产生的抗菌物质抑菌活性较强。     

Environmental Factors Affecting the Release and Dispersal of Ascospores of Mycosphaerella citri

ABSTRACT Greasy spot, caused by Mycosphaerella citri, produces a leaf spot disease affecting all citrus species in Florida and the Caribbean Basin. M. citri produces pseudothecia and ascospores, which are considered the principal source of inoculum, in decomposing leaves on the grove floor. In studies using a computer-controlled environmental chamber, a single rain event triggered release of most mature ascospores beginning 30 to 60 min after the rain event. Additional rain events did not bring about further release. High relative humidity without rain triggered release of low numbers of ascospores, but vibration and red/infrared irradiation had little or no effect on ascospore release. After three to four cycles of wetting and drying of leaves, all pseudothecia had matured and released their ascospores. In the field, ascospores were detectable starting about 2 h after the beginning of a rain or irrigation and most ascospores were released within 16 h. Ascospore release was greatest following rain events and somewhat less following irrigations, and low numbers of ascospores were detectable on days without precipitation. Ascospore numbers declined linearly with horizontal distance from the source and as a function of the logarithm of ascospore numbers with vertical distance. Low numbers of ascospores were detected 7.5 m above the ground and 90 m downwind from the grove. Ascospore release can be advanced by irrigating frequently during dry, nonconducive conditions to stimulate ascospore release when environmental conditions are unfavorable for infection, but the eventual effects on disease severity are uncertain.     

Mating and Pseudothecial Development in Mycosphaerella citri, the Cause of Citrus Greasy Spot

ABSTRACT Greasy spot, caused by Mycosphaerella citri, is a serious disease of citrus in the Caribbean basin. M. citri is a loculoascomycete and produces pseudothecia in decomposing leaves after intermittent wetting and drying. A new in vitro mating technique was developed for production of pseudothecia on sterilized leaf disks in petri dishes. Of the single-ascospore cultures that were recovered from individual asci, four were one mating type and four were a second mating type (tentatively designated mat+ and mat-), indicating that M. citri probably is heterothallic and bipolar like most other loculoascomycetes. Most populations of ascospores recovered from individual leaves or from leaves from groves of different citrus species and various locations had a 1:1 ratio of mating types consistent with random mating. Cytological studies demonstrated that the ontogeny of pseudothecial development was similar to other loculoascomycetes. The formation of mature pseudothecia required 30 to 45 cycles of wetting and drying of infected, dead leaves which required approximately 60 to 90 days. The in vitro system for pseudothecial production and the knowledge of the mating system in M. citri will facilitate genetic studies of this important pathogen.     

Genetic Differentiation and Host Specificity Among Populations of Alternaria spp. Causing Brown Spot of Grapefruit and Tangerine x Grapefruit Hybrids in Florida

ABSTRACT Alternaria spp. were sampled from brown spot lesions in several geographically separated citrus groves and different grapefruit and tangerine x grapefruit hybrid cultivars in Florida and screened for variation at 16 putative random amplified polymorphic DNA loci. Populations of the pathogen on two hybrids, Minneola and Orlando, in five locations throughout Florida were moderately differentiated (Nei's coefficient of gene differentiation [G(ST)] = 0.12) among locations. The hypothesis that host-specialized forms of Alternaria spp. cause brown spot on different Citrus spp. and cultivars was tested by estimating genetic differentiation among isolates sampled from different hosts and by pathogenicity assays. Isolates sampled from grapefruit and the hybrid cv. Nova were genetically distinct from isolates sampled from other hybrid cultivars including Robinson, Sunburst, Minneola, Orlando, and Murcott. No differentiation could be detected among isolates sampled from this latter group of hybrids. Quantitative pathogenicity assays on leaves using spray inoculation revealed that 'Nova' isolates were not significantly more pathogenic on 'Nova' compared with isolates from 'Minneola' and 'Orlando'. Similarly, grapefruit isolates were not significantly more pathogenic on grapefruit compared with isolates from 'Minneola'. Isolates from all hosts had similar disease rankings on each inoculated cultivar, with 'Minneola' the most susceptible, followed in decreasing order of susceptibility by 'Orlando', 'Sunburst', 'Nova', and 'Duncan' grapefruit. Rough lemon was generally immune to all isolates tested; however, occasional brown spot lesions were observed on leaves of this host with isolates from grapefruit. No evidence was found to support the hypothesis that unique genotypes of the pathogen, which are more virulent on 'Sunburst' or grapefruit, have been introduced to Florida. Populations of Alternaria spp. causing brown spot of citrus on grapefruit and 'Nova' in Florida are genetically distinct from isolates on other cultivars, and we speculate that these populations are in the early stages of adaptation to and possible speciation on these hosts.     

Effect of Modified Atmosphere for Control of Black Spot, Caused by Alternaria alternata, on Stored Persimmon Fruits

ABSTRACT Modified atmosphere packaging (MAP) of persimmon fruit resulted in the accumulation of acetaldehyde to a level of 80 mug/ml; ethanol to a level of 900 mug/ml; and CO(2) up to 30%. When fruits were stored at -1 degrees C for 4 months in such atmospheres, the incidence of black spot disease, caused by Alternaria alternata, was reduced. The effects of each of these gases were examined to determine their individual involvement in the inhibition of Alternaria development during storage. When A. alternata, grown at 20 degrees C on potato dextrose agar or inoculated in persimmon fruit, was exposed for 24 h to different levels of each volatile, acetaldehyde was the most fungistatic but only at concentrations higher than those that accumulated under MAP; CO(2) was moderately inhibitory at concentrations from 10 to 60%, whereas ethanol had no effect. Similar inhibitory effects were obtained with acetaldehyde at 620 mug/ml or 30% CO(2) when in vitro cultures of A. alternata and infected fruits were exposed for up to 2 weeks at 20 degrees C, but 1,000 mug of ethanol per ml had only a transitory inhibitory effect under these conditions. Based on analysis of the effect of concentration versus time for each gas accumulating in MAP, we suggest that the increasing concentration of CO(2) during storage is the principal factor in the inhibition of black spot disease development.     

链格孢菌的产毒培养条件及其毒素的致病范围

从世界性恶性杂草紫茎泽兰自然发生的叶斑病病斑上分离得到6个链格孢菌菌株,对它们的产毒情况进行了研究。筛选出产毒能力强的501菌株,确定了有利于该菌株产毒的温度为25℃、pH值为4．13、培养时间为5－7天、黑暗及静置等培养条件。选择74种植物,以离体叶片针刺法对粗毒素的致病范围进行了测试,表明粗毒素具有一定范围的选择作用特性,从中筛选出草莓、苦苣菜、鸭跖草、火柴头、刺槐、水花生等敏感植物作为毒素的生测材料。     

Factors Affecting the Release of Ascospores of Anisogramma anomala

ABSTRACT Relationships between environmental factors and release of ascospores of Anisogramma anomala, the causal agent of eastern filbert blight, were examined in four European hazelnut (Corylus avellana) orchards during a 2-year period. In each orchard, Burkhard volumetric spore traps and automated weather-monitoring equipment were deployed for 12-week periods beginning at budbreak, when hazelnut becomes susceptible to infection. Ascospores of A. anomala were released when stromata on the surface of hazelnut branches were wet from rain but not from dew. Release of ascospores ceased after branch surfaces dried. The duration of free moisture on branch surfaces regulated the initiation and rate of ascospore release, but no significant effects of temperature, relative humidity, wind, or light on ascospore release were apparent. Most (>90%) ascospores were captured during precipitation events that exceeded 20 h in duration, which represented about 10% of the total precipitation events each season. Quantitative relationships between the hourly capture of A. anomala ascospores and hours since the beginning of a precipitation event were developed. With the onset of precipitation, the hourly rate of ascospore capture increased until the fifth hour of rain, remained relatively constant between the fifth and twelfth hours, and then declined gradually. During the 12-week spore-trapping periods, the likelihood and rates of ascospore release associated with precipitation were highest at budbreak and then declined through April and May until early June, when the reserve of ascospores in the perithecia was depleted. Large numbers of ascospores were captured in the volumetric spore traps, indicating that ascospores may be commonly dispersed long distances on air currents as well as locally by splash dispersal within the canopy, as reported previously. The results indicate that monitoring seasonal precipitation patterns may be useful for estimating the quantity and temporal distribution of airborne inoculum during the period that the host is susceptible to infection.     

烟草赤星病颉颃放线菌的分离与鉴定

从健康小麦植株中分离得到21株分离物,经对7种植物病原菌颉颃试验分析,获得1株对烟草赤星病菌等具有良好颉颃活性的菌株2-7。采用平板对峙法、生长速率法、盆栽试验等方法测定其抑菌及生防作用。结果显示,放线菌2-7发酵液对烟草赤星病菌抑制率为62.1%,盆栽防治率为48.73%。通过该菌的培养特征、生理生化指标、细胞壁以及16S rDNA基因序列分析(GenBank登录号:JN638883),将菌株2-7归属于放线菌中的玫瑰暗黄链霉菌(Streptomyces roseofulvus)。     

枯草芽孢杆菌BL03对苹果霉心病和棉苗病害田间防治效果

枯草芽孢杆菌 (Bacillussubtilis) BL 0 3菌株 ,是作者从苹果树体上分离得到的拮抗菌株之一。室内测定对苹果霉心病菌 (Alternaria alternata)、炭疽病菌 (Colltotrichum gossypii)、红霉病菌 (Trichothecium roseum)和镰刀菌 (Fusarium sp.)等 1 0种病原菌的抑制效果均为1 0 0     

Genetic Analysis of Pathogenicity and Host-specific Toxin Production of Alternaria alternata Tomato Pathotype by Protoplast Fusion

Several pathotypes of Alternaria alternata are known to produce host-specific toxins (HSTs) as agents of pathogenicity or virulence. However, investigations into the genetic controls of HST biosynthesis and pathogenicity of Alternaria pathogens have been limited by the lack of a sexual stage in the life cycle of these pathogens. We report here the development of a protoplast fusion system and its use for genetic analysis of HST production and specific pathogenicity of the tomato pathotype of A. alternata that produces AAL-toxin as a HST. Drug-resistant transformants have been isolated by genetic transformation of nonpathogenic A. alternata (strain O-94) and A. alternata tomato pathotype (strain As-27) with vectors conferring resistance to hygromycin B and geneticin, respectively. Protoplasts of the respective transformants were fused by polyethylene glycol treatment or electrofusion. Fusion products were selected by culturing in the presence of both hygromycin B and geneticin, then confirmed by amplification using a polymerase chain reaction with specific primers to the transforming drug-resistance genes. Stable fusants were purified by successive subcultures on selective medium and single-spore isolation. The resultant stable fusants, probably inter-strain hybrids, had the same pathogenicity and toxin production as the wild-type strain As-27. These results suggest that protoplast fusion has potential applications for genetic analysis of A. alternata pathogens. Received 8 September 2000/ Accepted in revised form 27 October 2000     

UV-B辐射增强对链格孢菌(Alternaria alternata)生长、生理及致病力的影响

研究UV-B辐射增强对灯盏花(Erigeron breviscapus)叶斑病致病菌—链格孢菌(Alternaria alternata)的生长、生理及其致病力的影响,并探讨链格孢菌对UV-B辐射的响应机制。结果表明,UV-B辐射增强可延缓链格孢菌的生长,导致菌丝生长率、产孢量、纤维素酶活性及可溶性蛋白含量均显著降低(p0.05),链格孢菌的致病力显著下降(p0.05)。同一UV-B辐射强度下,20～60min的辐射处理对链格孢菌的生长、生理及致病力的影响没有显著性差异(p0.05)。链格孢菌表现出形态学和生理生化方面的UV-B适应机制,来减轻UV-B辐射的损伤。经UV-B辐射处理后的病菌积累较多黑色素,菌落颜色加深,菌丝分布更加致密,菌丝干重显著增加(p0.05);并且,链格孢菌的过氧化氢酶(CAT)活性显著升高(p0.05)。     

Laboratory Evaluation of Citrus Cultivars Susceptibility and Influence of Fruit Size on Fortune Mandarin to Infection by Alternaria alternata pv. citri

Young leaves of 62 citrus cultivars were inoculated with conidia of three Spanish isolates of Alternaria alternata pv. citri, the causal agent of brown spot of citrus. Hybrids with Dancy mandarin, King mandarin or their derivates as a parent, grapefruit cultivars and the mandarin cultivars Guillermina, Emperor, Clemenpons and Esbal were highly susceptible to the pathogen. Satsuma cultivar Clausellina and orange cultivars, with the exception of Sanguinelli, were slightly susceptible. Lemon and lime cultivars were not susceptible, with the exception of Mexican lime (Citrus aurantifolia), which was slightly susceptible. Although this study shows a range of potential hosts for this pathogen, to date the only affected cultivars in Spain are Fortune and Nova mandarins, and Minneola tangelo. The susceptibility of Fortune fruits decreased as diameter increased, being susceptible through the whole season. This was confirmed with field observations in autumn where fruit infections have been detected when the diameter reaches 6–7 cm.     

温度对烟草赤星病菌生物学特性及代谢表型的影响

为了解温度对烟草赤星病菌致病力及代谢表型的影响, 本研究采用菌丝生长速率法和离体叶片法, 分别测定不同温度下烟草赤星病菌的生长速率和致病力, 同时采用Biolog代谢表型技术测定了其在22?25?30℃和35℃下不同碳源?渗透压和pH下的代谢表型?结果表明, 烟草赤星病菌在15~35℃下均可生长, 30℃时菌丝生长最快?致病力和产孢能力最强, 35℃时孢子萌发率最高?在22?25?30℃和35℃时, 赤星病菌均可代谢Biolog FF代谢板上的95种碳源, 且随着温度升高对碳源的代谢能力逐渐增强?22℃和25℃时烟草赤星病菌对渗透压的适应范围最广, 其次为30℃, 35℃时适应范围最窄?在22?25?30℃和35℃时, 赤星病菌在pH 3.5~10范围内均可正常代谢, 在22?25℃和30℃下, 赤星病菌表现出强脱羧酶活性和弱脱氨酶活性, 在35℃下, 其脱羧酶和脱氨酶活性均相对较弱?研究结果揭示了烟草赤星病菌在不同温度下的适应能力, 为赤星病菌与环境互作研究提供了参考?