A tale of three acaropathogenic fungi in Israel: Hirsutella, Meira and Acaromyces

We review published and unpublished studies conducted in Israel with six acaropathogenic fungi, assayed in order to control the citrus rust mite, Phyllocoptruta oleivora (Ashmead) (CRM). Hirsutella thompsonii Fisher was introduced twice, killed 80-90% of the exposed mites, but due to its requirements for near-saturation humidities was deemed unsuitable for local outdoors conditions. Hirsutella kirchneri (Rostrup) Minter et al. and Hirsutella necatrix Minter et al. were also introduced and assayed against CRM and spider mites, but their efficacy was unsatisfactory. Three indigenous fungi found to be associated with mites, Meira geulakonigii, Meira argovae and Acaromyces ingoldii--all three recently described by Boekhout, Gerson, Scorzetti & Sztejnberg--were assayed against several mites. Meira geulakonigii killed 80-90% of several spider mites and of the CRM, and caused some mortality of Iphiseius degenerans (Berlese), one out of three phytoseiid predators assayed. Mortality was not due to parasitization; extracts from the media in which the fungi had developed caused considerable mite death, suggesting that it was a result of fungal toxins. Data from a field study indicated that spraying blastoconidia of M. geulakonigii on grapefruits infested by CRM significantly reduced pest-incurred damage from 23 to 13%. Applying qRT-PCR methodology indicated that M. geulakonigii was endophytic within sealed grapefruit flowers and in the flavedo of the fruits' peel. Neither in the laboratory nor in the field was any evidence ever obtained that this fungus damaged the plants, leading us to hypothesize that M. geulakonigii serves as a "body guard" of grapefruits (and perhaps other plants as well). All three fungi suffered very little mortality after being exposed to various insecticides and acaricides that are in current local use (with the exception of sulfur). The ability of M. geulakonigii to reduce mite numbers without affecting the host plant, the minimal fungal effect on some predatory mites, its endophytic nature along with the apparent tolerance of M. geulakonigii to many insecticides and acaricides, suggest that this fungus could be suitable for integrated pest management (IPM) program.     

毛竹上两株不同菌落形态的格氏梅拉菌Meira geulakonigii菌株的分离和特征记述

格氏梅拉菌Meira geulakonigii最早是在柑桔锈螨病的枯枝中发现的,它是一种担子菌类酵母样无性态真菌,归属于黑粉菌纲外担子菌亚纲。目前仅了解这种真菌在植物中的生长状态,关于它的宿主范围、区域分布和生长特性依然知之甚少。首次从竹子中分离了两株新的Meira geulakonigii菌株(PM1和PM2),并且更深一步地描述了他们的生理特性,尤其着重表述了菌丝体的独特生长现象。与先前报导的两株M.geulakonigii菌株PFS004和AS004相比较,PM1和PM2在分子系统上与AS004更相近,而生理现象与PFS004更相似。这两株菌株的成功分离可能有助于毛竹中Meira真菌菌丝体形成和功能的进一步研究。     

Diagnosis of Xanthomonas axonopodis pv. citri, causal agent of citrus canker, in commercial fruits by isolation and PCR-based methods

AIMS: To show the results of the detection of an EU quarantine organism, Xanthomonas axonopodis pv. citri (Xac), in citrus fruits imported from countries where this bacterium is present, using an integrated approach that includes isolation, pathogenicity assays and molecular techniques. METHODS AND RESULTS: Citrus fruits with canker-like symptoms, exported to Spain from South American countries were analysed by several methods. Bacterial isolation, three conventional polymerase chain reaction (PCR) protocols, and real-time PCR with SYBR Green or a TaqMan probe, were compared. Canker-like lesions were disrupted in PBS buffer, and the extract used for bacterial isolation and DNA extraction followed by PCR amplification. Canker lesions, identified by PCR, showed viable bacteria in eleven of fifteen fruit samples. In 16 out of 130 lesions analysed from these samples, Xac was isolated, and pathogenicity on grapefruit leaves confirmed. By real-time PCR, using SYBR green or a Taqman probe, Xac was detected in 58 and 80 lesions respectively. By conventional PCR the bacterium was detected in 39-52 lesions depending on the protocol employed. CONCLUSIONS: An integrated approach for reliable detection of Xac in lesions of fruit samples, employing several techniques and with real-time PCR using a TaqMan probe as the fastest and most sensitive screening method, has been established and validated and is proposed as a useful tool for the analysis of Xac on fresh fruits. SIGNIFICANCE AND IMPACT OF THE STUDY: This work faces up to the real threat of the importation of citrus fruits that can harbour quarantine bacteria and will be useful in diagnostic laboratories for the analysis of commercial fresh fruits from countries where citrus canker is present.     

Penicillin-Binding Proteins in Selenomonas ruminantium

Diphenyl, o-phenylphenol and thiabendazole were analyzed in citrus fruits. The peel and edible parts were separately homogenized. These fungicides were extracted with dichloromethane from the homogenate, and they were fractionated with Sephadex LH-20 columns. Gas chromatography was used to determine the presence of these fungicides. The fungicides found in edible parts of citrus fruits were confirmed by gas chromatography-mass spectrometry.

Diphenyl, o-phenylphenol and thiabendazole were detected in imported grapefruits, lemons and oranges. Almost all fungicides were found in the peel. The concentrations of the three fungicides in the edible parts were very low. Some samples contained all three fungicides in the edible parts.     

Sensitive and specific detection of Xanthomonas axonopodis pv. citri by PCR using pathovar specific primers based on hrpW gene sequences

A sensitive and specific assay was developed to detect citrus bacterial canker caused by Xanthomonas axonopodis pv. citri, in leaves and fruits of citrus. Primers XACF and XACR from hrpW homologous to pectate lyase, modifying the structure of pectin in plants, were used to amplify a 561 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally or artificially infected leaves of citrus. The PCR product was only produced from X. axonopodis pv. citri among 26 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference microbes.     

Citrus Leprosis and its Status in Florida and Texas: Past and Present

According to published reports from 1906 to 1968, leprosis nearly destroyed the Florida citrus industry prior to 1925. This was supported with photographs showing typical leprosis symptoms on citrus leaves, fruit, and twigs. Support for the past occurrence of citrus leprosis in Florida includes: (1) presence of twig lesions in affected orange blocks in addition to lesions on fruits and leaves and corresponding absence of similar lesions on grapefruit; (2) yield reduction and die-back on infected trees; and (3) spread of the disease between 1906 and 1925. Transmission electron microscopy (TEM) examination of tissue samples from leprosis-like injuries to orange and grapefruit leaves from Florida in 1997, and fruits from grapefruit and sweet orange varieties from Texas in 1999 and 2000 did not contain leprosis-like viral particles or viroplasm inclusions. In contrast, leprosis viroplasm inclusions were readily identified by TEM within green non-senescent tissues surrounding leprosis lesions in two of every three orange leaf samples and half of the fruit samples obtained from Piracicaba, Brazil. Symptoms of leprosis were not seen in any of the 24,555 orange trees examined across Florida during 2001 and 2002. The authors conclude that citrus leprosis no longer exists in Florida nor occurs in Texas citrus based on: (1) lack of leprosis symptoms on leaves, fruit, and twigs of sweet orange citrus varieties surveyed in Florida: (2) failure to find virus particles or viroplasm inclusion bodies in suspect samples from both Florida and Texas examined by TEM; (3) absence of documented reports by others on the presence of characteristic leprosis symptoms in Florida; (4) lack of its documented occurrence in dooryard trees or abandoned or minimal pesticide citrus orchard sites in Florida. In view of the serious threat to citrus in the U.S., every effort must be taken to quarantine the importation of both citrus and woody ornamental plants that serve as hosts for Brevipalpus phoenicis (Geijskes), B. californicus (Banks), and B. obovatus Donnadieu (Acari: Tenuipalpidae) from countries where citrus leprosis occurs.     

Larval <Emphasis Type="Italic">Cryptothelea gloverii</Emphasis> (Lepidoptera: Psycidae), an arthropod predator and herbivore on Florida citrus

The orange bagworm (OBW), Cryptothelea gloverii (Packard) (Lepidoptera: Psychidae) was previously reported feeding on citrus fruit and foliage and preying upon the camphor scale Pseudaonidia duplex (Cockerell) (Homoptera: Coccidae). In this study using laboratory assays, OBW preyed upon citrus rust mite, Phyllocoptruta oleivora (Ashmead) (Acari: Eriophyidae) and consumed eggs and adults of both P. oleivora and Panonychus citri (McGregor) (Acari: Tetranychidae), two important pest mites on Florida citrus. OBW was also observed to feed on the purple scale, Lepidosaphes beckii (Newman) (Homoptera: Diaspididae) and on a fungus (Penicillium sp.). OBW fed on orange and grapefruit leaves by starting from the border and eating part of the leaf, by chewing holes, or consuming the outer epithelium of either the axial or abaxial surface of the leaf without penetrating through the leaf. OBW was observed in orange orchards in association with fruit extensively russeted by P. oleivora feeding. Laboratory assays revealed that OBW larvae preferred to feed on oranges infested with P. oleivora rather than on clean fruits that were free of mite feeding damage. Feeding damage to citrus fruit by OBW larvae results in one to several holes being eaten into the rind or albedo, without damage to the fruit sacs.     

Assaying three new fungi against citrus mites in the laboratory,and a field trial

The Basidiomycotine fungi Meira geulakonigii, Meira argovae and Acaromyces ingoldii were assayed in the laboratory against five species of herbivorous mites: Phyllocoptruta oleivora (Eriophyidae), Panonychus citri, Eutetranychus orientalis, Tetranychus urticae and Tetranychus cinnabarinus (all four Tetranychidae). All fungi caused significantly high mortality rates (as compared to controls) after 14 days, some after 1 week. Phyllocoptruta oleivora was the most susceptible, showing >80% mortality even after 1 week. In a field trial, grapefruits sprayed either once a month or once a season with M. geulakonigii had significantly fewer P. oleivora and less damage than unsprayed fruit. These results suggest that M. geulakonigii may protect grapefruits against the injurious P. oleivora.     

Evidence for host plant preference by Iphiseiodes quadripilis (Acari: Phytoseiidae) on Citrus

In this study, we present field and laboratory evidence on the preference of Iphiseiodes quadripilis (Banks) for grapefruit (Citrus paradisi Macfadyen) leaves compared with sweet orange (Citrus sinensis (L.) Osbeck) leaves. This preference was confirmed in four orchards whether leaf samples were taken from either border trees of contiguous grapefruit or sweet orange or interior row trees with both citrus species in adjacent rows. Iphiseiodes quadripilis was most abundant in grapefruit trees in spite of the greater abundance of the Texas citrus mite, Eutetranychus banksi (McGregor) (Acari: Tetranychidae) in sweet orange trees. Similar preference responses were observed in laboratory tests using a Y-tube olfactometer whether I. quadripilis were collected from sweet orange or grapefruit. Iphiseiodes quadripilis collected from grapefruit trees showed significant preference for grapefruit over sweet orange leaves in contact choice tests using an arena of alternating leaf strips (12 mm long × 2 mm wide) of sweet orange and grapefruit. However, I.␣quadripilis collected from sweet orange trees did not show preference for either grapefruit or sweet orange leaves. Based on these results, grapefruit leaves foster some unknown factor or factors that retain I. quadripilis in greater numbers compared with sweet orange leaves.     

Terpene down-regulation in orange reveals the role of fruit aromas in mediating interactions with insect herbivores and pathogens

Plants use volatile terpene compounds as odor cues for communicating with the environment. Fleshy fruits are particularly rich in volatiles that deter herbivores and attract seed dispersal agents. We have investigated how terpenes in citrus fruit peels affect the interaction between the plant, insects, and microorganisms. Because limonene represents up to 97% of the total volatiles in orange (Citrus sinensis) fruit peel, we chose to down-regulate the expression of a limonene synthase gene in orange plants by introducing an antisense construct of this gene. Transgenic fruits showed reduced accumulation of limonene in the peel. When these fruits were challenged with either the fungus Penicillium digitatum or with the bacterium Xanthomonas citri subsp. citri, they showed marked resistance against these pathogens that were unable to infect the peel tissues. Moreover, males of the citrus pest medfly (Ceratitis capitata) were less attracted to low limonene-expressing fruits than to control fruits. These results indicate that limonene accumulation in the peel of citrus fruit appears to be involved in the successful trophic interaction between fruits, insects, and microorganisms. Terpene down-regulation might be a strategy to generate broad-spectrum resistance against pests and pathogens in fleshy fruits from economically important crops. In addition, terpene engineering may be important for studying the basic ecological interactions between fruits, herbivores, and pathogens.     

Brown spot disease of citrus caused by Phaeoisariopsis sp.

Brown spot disease of Citrus spp. was shown to be caused by Phaeoisariopsis sp. It severely affects all varieties of sweet orange, tangerine, grapefruit, lime and rough lemon, smooth lemon being relatively resistant. Fruits and leaves are much more susceptible than stems on which symptoms are rare. On the young fruit circular, slightly sunken, brown necrotic lesions are often associated with a surrounding ring of raised epicarp, giving the fruit a blistered appearance. Lesions on older fruits are usually flat and brown and surrounded by yellow haloes. Premature abscission, especially of young fruits, is common. Leaf symptoms start as greenish yellow patches and a fully formed leaf spot consists of light brown or greyish centre which is surrounded by a dark brown margin bordered by a yellow halo. Generalised foliar chlorosis, caused by coalescence of several lesions, culminates in premature defoliation. During wet weather, centres of both fruit and foliar lesions sporulate and become black. Stem lesions, dark brown and mostly occurring as extensions of petiole lesions, may coalesce causing stem die-back or resulting in the formation of corky internodal lesions. In artificial culture, the fungus sporulated only on media containing an extract of citrus leaf or fruit peel. The characteristics of the fungus mostly based on examination of structures formed on naturally infected parts of the plant, are described. This is probably the first report of a Phaeoisariopsis sp. on citrus.     

Larval Cryptothelea gloverii (Lepidoptera: Psycidae), an arthropod predator and herbivore on Florida citrus

The orange bagworm (OBW), Cryptothelea gloverii (Packard) (Lepidoptera: Psychidae) was previously reported feeding on citrus fruit and foliage and preying upon the camphor scale Pseudaonidia duplex (Cockerell) (Homoptera: Coccidae). In this study using laboratory assays, OBW preyed upon citrus rust mite, Phyllocoptruta oleivora (Ashmead) (Acari: Eriophyidae) and consumed eggs and adults of both P. oleivora and Panonychus citri (McGregor) (Acari: Tetranychidae), two important pest mites on Florida citrus. OBW was also observed to feed on the purple scale, Lepidosaphes beckii (Newman) (Homoptera: Diaspididae) and on a fungus (Penicillium sp.). OBW fed on orange and grapefruit leaves by starting from the border and eating part of the leaf, by chewing holes, or consuming the outer epithelium of either the axial or abaxial surface of the leaf without penetrating through the leaf. OBW was observed in orange orchards in association with fruit extensively russeted by P. oleivora feeding. Laboratory assays revealed that OBW larvae preferred to feed on oranges infested with P. oleivora rather than on clean fruits that were free of mite feeding damage. Feeding damage to citrus fruit by OBW larvae results in one to several holes being eaten into the rind or albedo, without damage to the fruit sacs.     

A TaqMan PCR Method for Routine Diagnosis of the Quarantine Fungus Guignardia citricarpa on Citrus Fruit

With respect to disease risk for the quarantine fungus Guignardia citricarpa on citrus fruit an accurate diagnosis for routine analysis is required. Also, when inspections have to be performed on imported citrus fruits, a fast detection method is urgently needed. A fast automated DNA extraction method based on magnetic beads combined with a real‐time PCR assay was optimized to improve and advance the routine diagnosis of citrus black spot disease. Real‐time PCR was used for detection of the pathogen G. citricarpa in planta. A specific primer/TaqMan probe combination that discriminates between G. citricarpa and the harmless citrus endophyte Guignardia mangiferae, was designed based on the internal transcribed spacer region of the multi‐copy rDNA gene. Co‐amplification of target DNA along with an internal competitor DNA fragment made the diagnostic assay more reliable to check for false negatives. The real‐time PCR was specific, since no cross reaction was observed with a series of citrus pathogens and related species. The diagnostic assay was performed on lesions dissected from imported diseased oranges. Comparison between the conventional PCR and the real‐time PCR methods showed that the TaqMan method was more sensitive.     

Hirsutella thompsonii a fungal parasite of the citrus rust mitePhyllocoptruta oleivora in the Rio Grande Valley of Texas

Hirsutella thompsonii, a hyphomycetous fungus, was found attacking the citrus rust mitePhyllocoptruta oleivora for the first time in May, 1972, in the Lower Rio Grande Valley of Texas. Sharp reductions in mite populations occurred during this period. The fungus was isolated from mites collected from citrus fruits and leaves. The parasitic nature and the distribution of the pathogen are described.     

Are citrus species favorable hosts for the Mediterranean fruit fly? A demographic perspective

We studied, under laboratory conditions, demographic parameters of adult Mediterranean fruit flies, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) (medfly), obtained from three sweet orange varieties, lemon, and bitter oranges. These data were combined with immature developmental rates and survival on the same hosts to estimate host‐specific population parameters. Pairs of newly emerged adults from each citrus variety were held individually in transparent plastic cages, and females were allowed to oviposit in either red domes (artificial, pre‐punctured plastic oviposition devices), or intact, whole citrus fruits. We found strong effects of larval host (citrus fruits) on adult longevity and fecundity. In all five citrus varieties, females did not manage to deposit eggs into fruit pulp. The proportion of eggs laid in either the flavedo or albedo area of the fruit peel differed depending on the citrus variety. In all cases except bitter oranges, females oviposited fewer eggs in citrus fruits than in the artificial oviposition substrates, suggesting that most citrus fruits cause a significant reduction in the reproductive potential of medflies. Negative correlations were found between fecundity and (a) the density of oil glands, and (b) the amount of essential oils in the flavedo area of citrus fruits. There was no correlation between fecundity and other fruit physical characteristics, such as resistance of fruit peel to pressure and thickness of the flavedo. Apparently, resistance of citrus fruits to medfly infestation is directly related to citrus essential oils. The intrinsic rate of increase (r) was higher in bitter oranges than in the three sweet orange varieties tested. A negative r was estimated for flies that developed and oviposited in lemons, indicating a tendency for population decrease in this host. The suitability of citrus fruits for medfly development and the practical implications of our findings for management of medflies in citrus orchards are discussed.     

Argovin,a novel natural product secreted by the fungus Meira argovae,is antagonistic to mites

A metabolite of the fungus Meira argovae Boekhout, Scorzetti, Gerson & Sztejnberg (Exobasidiomycetidae) was assayed as an antagonist of mites. Separation of extracted fungal metabolites by reversed phase liquid chromatography (RPLC), with subsequent testing of the obtained fractions, allowed us to isolate a single mite‐antagonistic fraction (also active against a bacterium) that primarily includes one major component. This active compound (herein termed ‘argovin’) was identified by analyzing its spectral characteristics as 4,5‐dihydroxyindan‐1‐one, which has previously only been described as a product of chemical reactions. The growth rate of the fungus was higher at a neutral pH than at an acidic one. Meira argovae adjusts the pH of its media to values optimal for its colony growth and toxic secretions. RPLC‐cleaned argovin at 0.2 mg ml^?1 killed 100% of a population of the citrus rust mite, Phyllocoptruta oleivora (Ashmead) (Acari: Eriophyidae). This trait may be used to control citrus rust mites in the field, as well as for toxin production for industrial and pharmaceutical uses.     

Brevipalpus californicus, B. obovatus, B. phoenicis, and B. lewisi (Acari: Tenuipalpidae): a Review of their Biology, Feeding Injury and Economic Importance

The genus Brevipalpus includes most of the economically important species of Tenuipalpidae. Many Brevipalpus species reproduce by theletokous parthenogenesis while other species reproduce by male fertilization of female eggs. Previous researchers have determined that Brevipalpus californicus (Banks), B. obovatus Donnadieu, and B. phoenicis (Geijskes) females were haploid with two chromosomes. The life cycle and developmental times for these three species are reviewed. Longevity of each Brevipalpus species is two to three times greater than corresponding longevities of various tetranychid mites. Brevipalpus mites inject toxic saliva into fruits, leaves, stems, twigs, and bud tissues of numerous plants including citrus. Feeding injury symptoms on selected plants include: chlorosis, blistering, bronzing, or necrotic areas on leaves by one or more Brevipalpus mites. Premature leaf drop occurred on 'Robinson' tangerine leaves in Florida (USA). Leaf drop was observed in several sweet orange and grapefruit orchards in Texas (USA) that were heavily infested with Brevipalpus mites feeding on the twigs, leaves, and fruit. Initial circular chlorotic areas appear on both sweet orange and grapefruit varieties in association with developing populations of Brevipalpus mites in Texas. These feeding sites become progressively necrotic, darker in color, and eventually develop into irregular scab-like lesions on affected fruit. Russeting and cracking of the fruits of other plant hosts are reported. Stunting of leaves and the development of Brevipalpus galls on terminal buds were recorded on sour orange, Citrus aurantium L., seedlings heavily infested with B. californicus in an insectary. The most significant threat posed by these mites is as vectors of a potentially invasive viral disease called citrus leprosis.     

Antioxidative properties of Jaffa sweeties and grapefruit and their influence on lipid metabolism and plasma antioxidative potential in rats

The effective substances (polyphenols, phenolic and ascorbic acids, flavonoids and dietary fibers) and antioxidative activities, using different radical-scavenging tests, were determined for Jaffa sweeties and grapefruit. The antioxidative activities comprised the contributions from polyphenols, phenolic acids, flavonoids and ascorbate components, and were well-correlated with polyphenols and flavonoids. The correlation coefficient between the polyphenols and antioxidative activity varied from 0.73 to 0.99. All applied methods showed that sweeties had higher antioxidative activity than grapefruit. Experiments on laboratory animals show that diets supplemented with sweeties, and to a lesser extent with grapefruit, increased the plasma antioxidative potential and improved the lipid metabolism, especially in the rats fed with added cholesterol. These findings provide additional characterization of the nutritional value of citrus fruits and their influence on the lipid metabolism in rats.     

Botanical insecticides in controlling Kelly's citrus thrips (Thysanoptera: Thripidae) on organic grapefruits

Kelly's citrus thrips, Pezothrips kellyanus (Bagnall) (Thysanoptera: Thripidae) was first recorded in Cyprus in 1996 and became an economic citrus pest. In Cyprus, Kelly's citrus thrips larvae cause feeding damage mainly on immature lemon and grapefruit fruits. Use of botanical insecticides is considered an alternative tool compared with synthetic chemicals, in offering solutions for healthy and sustainable citrus production. During 2008-2010, the efficacy of the botanical insecticides azadirachtin (Neemex 0.3%W/W and Oikos 10 EC), garlic extract (Alsa), and pyrethrins (Vioryl 5%SC) was evaluated in field trials against Kelly's citrus thrips larval stage I and II aiming at controlling the pest's population and damage to organic grapefruit fruits. In each of the trial years treatments with pyrethrins and azadirachtin (Neemex 0.3%W/W) were the most effective against Kelly's citrus thrips compared with the untreated control (for 2008: P < 0.018; for 2009: P < 0.000; for 2010: P < 0.008). In 2008, the mean number of damaged fruits in treatments with pyrethrins and Neemex was 9.6 (19.2%) and 9.7 (19.5%) respectively, compared with 12.2 (24.3%) in the untreated control. In 2009, the mean number of damaged fruits in treatment with pyrethrins was 3.7 (7.3%) and 3.9 (7.8%) in treatment with Neemex compared with 8.6 (17.3%) in the untreated control, while in 2010 the mean damaged fruits in these treatments was recorded at 18.7 (37.5%) and 19.6 (39.2), respectively, compared with 29.6 fruits (59.2%) in the control. Oikos 10 EC showed significant effect only in 2009 and 2010. In these years, the mean number of damaged fruits was recorded at 5.5 and 21.2 compared with 8.6 and 29.6 fruits in the untreated control, respectively. Garlic extract showed the lowest effect from all the botanicals used compared with the untreated control.