Raghuwinder Singh

Laboratory And Field Evaluations Of Silwet L-77 And Kinetic Alone and in Combination with Imidacloprid and Abamectin for the Management of the Asian Citrus Psyllid, Diaphorina Citri (Hemiptera: Psyllidae)

Abstract Silwet L-77, an organosilicone adjuvant, was used to enhance coverage of an entomopathogenic fungus in field trials conducted in a central Florida citrus research grove. The results indicated that Silwet L-77, by itself, was toxic to nymphs of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). Laboratory bioassays were conducted to confirm the toxicity of the adjuvant to eggs, nymphs, and adults of D. citri. Silwet L-77 at 0.05% (500 ppm) killed all nymphs, but was not as effective against eggs and adults. However, Silwet L-77, when combined with a reduced rate (one-tenth the lowest label rate = LLR) of imidacloprid, killed >90% of eggs and adults in laboratory tests. In a subsequent field trial, the combination of Silwet L-77 and one-tenth the LLR of imidacloprid gave good control of nymphs, but exhibited weak residual toxicity to adults when compared to imidacloprid at the LLR. Additional trials were then conducted with Silwet L-77 and Kinetic, another organosilicone adjuvant, alone and in combination with different rates of imidacloprid or abamectin using potted citrus trees in the greenhouse. Combining Silwet L-77 or Kinetic with one-fourth or one-half the LLR of imidacloprid killed as many eggs, nymphs, or adults as the LLR of imidacloprid. The combination of Silwet L-77 or Kinetic with one-fourth or one-half the LLR of abamectin killed as many eggs and nymphs as the LLR, but did not control adults as well. The results are discussed in terms of managing the Asian citrus psyllid in Florida now that citrus greening disease is endemic.

Evaluations of a Novel Isolate of Isaria fumosorosea for Control of the Asian Citrus Psyllid, Diaphorina citri (Hemiptera: Psyllidae)

ABSTRACT A fungal pathogen that killed adult Diaphorina citri Kuwayama (Asian citrus psyllid) in Florida citrus groves was isolated, characterized molecularly and morphologically and identified as a novel isolate of Isaria fumosorosea (Ifr) (= Paecilomyces fumosoroseus) from the Asian citrus psyllid (Ifr AsCP), but no concentration-mortality or time-response data were obtained. When adult psyllids were sprayed with spores at 28°C, time response (LT50) values of 111 and 102.5 h at spore concentrations of 1 × 107 and 1 × 108 spores/mL, respectively, were obtained. The LT99, was 167.4 and 174.6 h, respectively, for the 2 spore concentrations. After 192 h, the LC50 value was 6.8 × 105 spores/mL and the LC99 was 2.2 × 108 spores/mL. Ten serial passages Ifr AsCP were carried out on malt extract agar (MEA) and dilute Sabouraud dextrose agar and yeast (SDY) media. The pathogenicity to adult psyllids did not decline, but spore yield declined on the SDY medium and Ifr AsCP consistently produced more spores on SDY than on MEA media. Ifr AsCP was highly pathogenic to the psyllid when healthy adults were exposed to spores from psyllid cadavers stored at -74°C. Two pilot field trials were conducted in Florida citrus groves to assess methods for confirming infection; Ifr AsCP spores infected immature psyllids when applied at a rate of 1 × 107 spores/mL, but monitoring for infected nymphs required special handling methods.

CITRUS LEAFMINER, PHYLLOCNISTIS CITRELLA (LEPIDOPTERA: GRACILLARIIDAE), AND NATURAL ENEMY DYNAMICS IN CENTRAL FLORIDA DURING 2005

Abstract After the citrus leafminer (CLM), Phyllocnistis citrella Stainton (Lepidoptera: Gracillariidae), invaded Florida in 1993, the endoparasitoid Ageniaspis citricola Logvinovskaya (Hymenoptera: Encyrtidae) was introduced in 1994 in a classical biological control program. Subsequent to its establishment, only limited information has been obtained regarding the seasonal abundance of A. citricola and its host in central Florida citrus groves. During 2005, we monitored replicated plots treated with oil or imidacloprid once on 23 Jun 2005, along with untreated control trees, in a Polk County commercial Valencia orange grove on a weekly basis when tender new growth (= flush) was available. As expected, CLM abundance in the early spring flush was nearly undetectable due to the lack of suitable flush during winter when CLM populations decline nearly to zero. Also as expected, A. citricola was not found during this time. During the second flush (Jun through Jul) CLM populations increased and A. citricola appeared, parasitizing up to 39% of the pupae in the untreated controls and up to 33% in the blocks treated with oil. Imidacloprid did not significantly reduce the number of CLM larvae but did reduce Asian citrus psyllid, Diaphorina citri Kuwayama, nymphal densities. Peak abundance of the CLM occurred during the third flush cycle on 5 Oct from trees treated once with oil, with a mean (SD) of 1.3 (0.8) CLM mines per leaf. Parasitism by A. citricola increased through the season, peaking at 56% of the CLM that had pupated prior to the 16 and 23 Nov samples in the untreated control trees and at 37% in the oil-treated trees; A. citricola was not found in imidacloprid-treated trees on those dates. During the growing season, a high proportion (up to 100% in some samples) of the CLM mines were empty, presumably due to predation. The data confirmed, for the first time, that A. citricola is an important natural enemy of those CLM larvae that escaped predation in this citrus-growing area in Florida. Nymphs of the Asian citrus psyllid were significantly reduced for 3 weeks after the imidacloprid treatment. However, shoots on trees treated with imidacloprid were significantly shorter than shoots on untreated trees and the number of shoots produced in imidacloprid-treated trees was reduced, raising concerns that imidacloprid might affect growth of citrus flush. Brown citrus aphids were nearly absent throughout the growing season.

LOW INCIDENCE OF CANDIDATUS LIBERIBACTER ASIATICUS IN DIAPHORINA CITRI (HEMIPTERA: PSYLLIDAE) POPULATIONS BETWEEN NOV 2005 AND JAN 2006: RELEVANCE TO MANAGEMENT OF CITRUS GREENING DISEASE IN FLORIDA

Citrus greening disease or Huanglongbing (HLB) is caused by the gram-negative bacterium Candidatus Liberibacter asiaticus (Ca. L. asiaticus) (Garnier et al. 2000) and was confirmed in southern Florida in 2005 (Halbert 2005; Bouffard 2006). This disease is vectored by Diaphorina citri Kuwayama (Hemiptera: Psyllidae), which colonized the citrus-growing regions of Florida after it was discovered in 1998 (Knapp et al. 1998; Halbert 1998; Halbert et al. 2000). Diaphorina citri acquires the greening bacterium while feeding on infected phloem (Hung et al. 2004). HLB ultimately is fatal to susceptible citrus trees, so early detection and removal of infected trees is important for disease management. Unfortunately, citrus trees often are asymptomatic for years before the common signs of HLB, including yellowing and mottling of leaf veins and misshapen green-colored fruit, are noticeable (da Graga 1991). Current chemical and biological controls reduce D. citri populations (Rae et al. 1997; Hoy et al. 1999; Hoy & Nguyen 2000; Michaud 2004; Browning et al. 2006), but may not be sufficient to eliminate all HLB transmission. It will be important to understand the epidemiology of HLB to control the spread of this disease. The regions of Florida with citrus showing symptoms of HLB currently are being mapped (http://www.doacs.state.fl.us/pi/chrp/greening/maps/ cgsit_map.pdf). However, little currently is known about infection rates and transmission

Detection and identification of Clerodendron golden mosaic China virus in Salvia splendens

Two viral DNAs were obtained by rolling circle amplification conducted on DNA extracts from salvia (Salvia splendens) cv. ‘Dancing Flame’ showing variegated foliage resembling virus symptoms. Sequence analyses revealed the presence of an isolate of the putative begomovirus Clerodendron golden mosaic China virus. The virus was detected in all salvia plants showing variegated foliage, but not in non-variegated plants. The virus was graft-transmitted to healthy salvias which reproduced the original symptoms. Attempts to transmit the virus with the whitefly Bemisia tabaci biotype B failed. The results of this investigation strongly suggest that the salvia isolate of Clerodendron golden mosaic China virus (ClGMCNV-Sal[USA:LA:11]) is the cause of the variegated foliage of S. splendens cv. ‘Dancing Flame’. This is the first report of this virus in the United States.

A TaqMan-based real time PCR assay for specific detection and quantification of Xylella fastidiosa strains causing bacterial leaf scorch in oleander.

A TaqMan-based real-time PCR assay was developed for specific detection of strains of X. fastidiosa causing oleander leaf scorch. The assay uses primers WG-OLS-F1 and WG-OLS-R1 and the fluorescent probe WG-OLS-P1, designed based on unique sequences found only in the genome of oleander strain Ann1. The assay is specific, allowing detection of only oleander-infecting strains, not other strains of X. fastidiosa nor other plant-associated bacteria tested. The assay is also sensitive, with a detection limit of 10.4fg DNA of X. fastidiosa per reaction in vitro and in planta. The assay can also be applied to detect low numbers of X. fastidiosa in insect samples, or further developed into a multiplex real-time PCR assay to simultaneously detect and distinguish diverse strains of X. fastidiosa that may occupy the same hosts or insect vectors. Specific and sensitive detection and quantification of oleander strains of X. fastidiosa should be useful for disease diagnosis, epidemiological studies, management of oleander leaf scorch disease, and resistance screening for oleander shrubs.

First report of Alternaria alternata causing leaf spot on Aloe vera in Louisiana.

Aloe vera (L.) Burm. f. is a perennial succulent plant that is grown worldwide mainly for medicinal and cosmetic uses. In the USA, it is mainly cultivated in some southern states to produce aloe gel for the cosmetic industry (3), and in Louisiana it is also sold commercially as an ornamental. During the summer of 2011, several A. vera plants infected with leaf spots were observed on the campus of Louisiana State University, Baton Rouge. Large, necrotic, sunken, circular to oval, dark brown spots were present on both surfaces of the leaves. Infected leaf tissue pieces were surface disinfested with 1% NaOCl solution for 1 min and plated on potato dextrose agar (PDA). Plates were incubated at 28°C in the dark for 4 days. A dark olivaceous fungus with profuse golden brown, branched, and septate hyphae was consistently isolated from the infected tissue on PDA. The fungus produced conidia with longitudinal and transverse septa, and was morphologically identified as an Alternaria sp. (4). Conidia were produced in long chains, pale to light brown, obpyriform, with a beak (6.0 μm long), one to seven transverse and up to three longitudinal septa, and measured 10 to 45 μm long × 7 to 18 μm wide. Conidiophores were straight, septate, light to olive golden brown with conidial scar, and measured 35 to 100 μm long × 2 to 5 μm wide. Genomic DNA from a single-spored isolate was extracted and the internal transcribed spacer (ITS1-5.8s-ITS2) regions were amplified and sequenced using primers ITS1 and ITS4. BLASTn analysis of a 486-bp sequence (GenBank Accession No. JQ409455) resulted in 100% homology with A. alternata strain DHMJ16 (GenBank Accession No. JN986768) from China and several other Alternaria spp. The fungus was identified as A. alternata based on mycelial and conidia characters after being grown under standard, previously described conditions (4). Pathogenicity tests were carried out by inoculating six potted aloe plants with 0.5-cm diameter discs taken from a 6-day-old culture grown on PDA. Four discs were placed on the upper surface of each of the bottom leaves of every plant. Inoculated plants were individually covered with a plastic bag and maintained in a greenhouse for 1 week at 25 ± 2°C. Six control plants received only agar plugs. Seven days after inoculation, necrotic leaf spots were observed on the inoculated plants and A. alternata was reisolated from these spots. No leaf spots were observed on control plants. To the best of our knowledge, this is the first report of leaf spot caused by A. alternata on A. vera in Louisiana. Several outbreaks of the disease have been reported in Pakistan and India as damaging aloe gel production in those countries (1,2). An outbreak of this disease in Louisiana could represent a serious issue for the states A. vera ornamental commerce. References: (1) R. Bajwa et al. Can. J. Plant Pathol. 32:490, 2010. (2) A. Kamalakannan et al. Australas. Plant Dis. Notes 3:110, 2008. (3) T. Reynolds. Aloes: The Genus Aloe. CRC Press, Boca Raton, FL, 2004. (4) E. G. Simmons. Alternaria: An Identification Manual: Fully Illustrated and with Catalogue Raisonné 1796-2007. CBS Fungal Biodiversity Centre, Utrecht, The Netherlands, 2007.

First Report of Xylella fastidiosa Associated with Oleander Leaf Scorch in Louisiana

Oleander (Nerium oleander L.) is an evergreen shrub native to the Mediterranean Region and Southeast Asia. Despite being poisonous, it is a popular ornamental plant for use in landscapes, gardens, parks, roadsides, and highway medians. During the fall of 2008, several oleander plants with leaf scorch symptoms were observed at Arsenal Park in Baton Rouge, LA. Symptomatic oleander samples were also received from a commercial nursery in Baton Rouge, LA and a homeowner in Thibodeaux, LA. Symptoms resembled leaf scorch caused by Xylella fastidiosa Wells et al. and included chlorotic mottling of the leaves that started from the tips and margins and progressed toward the midribs. As disease developed, leaf tips and margins became necrotic. Severely infected plants defoliated and died. Leaf petioles from 13 samples (8 from Arsenal Park, 3 from the commercial nursery, and 2 from the homeowner) from symptomatic plants gave positive reactions for X. fastidiosa by ELISA (Agdia, Inc., Elkhart, IN). Leaf petioles from six healthy oleander plants gave a negative reaction for X. fastidiosa by ELISA. Isolation of X. fastidiosa was attempted from eight ELISA-positive and six ELISA-negative oleander samples. Leaf petioles weighing 0.05 g from each sample were used for isolation. The petioles were surface sterilized in 70% ethanol for 1.5 min and then in 2% sodium hypochlorite for 1.5 min, followed by three 1-min washes in sterile water. The petioles were chopped into small pieces under aseptic conditions and soaked in 500 μl sterile water for 30 min. One hundred microliters of the suspension were spread onto periwinkle wilt (PW) plates and incubated in the dark at 28°C. After incubation for 7 days, bacterial colonies typical of X. fastidiosa appeared on five of eight ELISA-positive sample plates. No colonies were observed on six ELISA-negative sample plates. Single colonies were transferred to fresh PW plates to obtain pure cultures. Bacterial colonies from five pure cultures were suspended in nuclease-free water and boiled for 10 min to obtain DNA. DNA from eight symptomatic and six healthy oleander plants was extracted with a DNeasy Plant Mini kit (Qiagen Inc., Valencia, CA) according to the manufacturers guidelines. Primers (QHOLS-08 and QHOLS-05) (1) specific to the oleander strain of X. fastidiosa amplified a 274-bp portion of DNA from both symptomatic oleander tissues and pure culture of X. fastidiosa isolated from symptomatic tissue. No such amplification was observed in healthy tissue. These primers amplify a portion of DNA encoding a hypothetical protein of unknown function that has been shown to be unique to oleander strains of X. fastidiosa. The PCR product was sequenced and compared with the whole genome shotgun sequence of the oleander strain Ann-1 of X. fastidiosa (GenBank Accession No. AAAM03000099), which resulted in 100% identity with nucleotides 11343 to 11616 in contig 228. X. fastidiosa has been previously reported to cause oleander leaf scorch in California (3), Florida (4), and Texas (2). To our knowledge, this is the first report of X. fastidiosa associated with oleander leaf scorch in Louisiana, extending the geographic range of this important bacterial disease. References: (1) Q. Huang. Curr. Microbiol. 58:393, 2009. (2) Q. Huang et al. Plant Dis. 88:1049, 2004. (3) A. H. Purcell et al. Phytopathology 89:53, 1999. (4) R. L. Wichman et al. Plant Dis. 84:198, 2000.

TOOLS FOR EVALUATING LIPOLEXIS OREGMAE (HYMENOPTERA: APHIDIIDAE) IN THE FIELD: EFFECTS OF HOST APHID AND HOST PLANT ON MUMMY LOCATION AND COLOR PLUS IMPROVED METHODS FOR OBTAINING ADULTS

Abstract Lipolexis oregmae Gahan was introduced into Florida in a classical biological control program directed against the brown citrus aphid, Toxoptera citricida (Kirkaldy), on citrus. Prior to evaluating distribution, host range, and potential nontarget effects of L. oregmae in Florida, we evaluated the role of other potential host aphids and host plants on mummy production and location. Under laboratory conditions, this parasitoid produced the most progeny on the target pest, the brown citrus aphid on citrus. This parasitoid, unlike the majority of aphidiids, did not produce mummies on any of the host plants tested when reared in black citrus aphid T. aurantii (Boyer de Fonscolombe) on grapefruit, spirea aphid Aphis spiraecola Patch on grapefruit and pittosporum, cowpea aphid A. craccivora Koch on grapefruit and cowpeas, or melon aphid A. gossypii Glover on grapefruit and cucumber. Thus, sampling for L. oregmae mummies of these host aphids and host plants must involve holding foliage in the laboratory until mummies are produced. This parasitoid requires high relative humidity to produce adults because no adults emerged when mummies were held in gelatin capsules, but high rates of emergence were observed when mummies were held on 1.5% agar plates. In addition, we compared the color of 6 aphid hosts and the color of mummies produced by L. oregmae when reared in them to determine if color of mummies could be used to identify L. oregmae. Mummy color varied between aphid hosts and tested host plants, and is not a useful tool for identifying L. oregmae for nontarget effects.

First report of Fusarium wilt of Canary Island date palm caused by Fusarium oxysporum f. sp. canariensis in Louisiana.

Canary Island date palm (Phoenix canariensis Hort. Ex Chabaud) is a signature palm planted in New Orleans, LA. Currently, the city has approximately 1,000 mature Canary Island date palms. During the fall of 2009, 153 palms were inspected with 27 palms exhibiting typical symptoms of Fusarium wilt. Symptoms included one-sided death and a reddish brown streak on the rachis of affected fronds and death of the leaflets. Longitudinal sections of affected fronds showed vascular discoloration. Severely infected palms were completely dead. Small pieces of diseased tissue from five palms were surface sterilized with sodium hypochlorite (0.6%) for 2 to 3 min, then rinsed in sterile distilled water, blotted dry, and plated on potato dextrose agar (PDA). Fungal colonies on PDA produced a purple pigment, and both macro- and microconidia that are typical of Fusarium oxysporum were observed under a light microscope. A single-spore culture of isolate PDC-4701 was obtained. DNA from this isolate was extracted with a DNeasy Plant Mini kit (Qiagen Inc., Valencia, CA) and primers ef1 and ef2 were used to amplify and sequence the translation elongation factor 1-α gene (2). NCBI BLAST analysis of the 616-bp sequence resulted in 100% identity with F. oxysporum f. sp. canariensis isolates PLM-385B from Texas and PLM-511A from South Carolina (GenBank Accession Nos. HM 591538 and HM 591537, respectively). Isolate PDC-4701, grown on PDA for 2 weeks, was used to inoculate 10 9-month-old P. canariensis seedlings. An 18-gauge needle was used to inject 15 ml of a 107 conidia/ml suspension into the stem near the soil line. Each seedling was inoculated at two locations and covered with Parafilm at the inoculation sites. Ten control seedlings were injected with sterile distilled water in the same manner. Inoculated and control seedlings were maintained in a greenhouse at 28 ± 2°C. Leaves of all 10 inoculated seedlings started to wilt 3 months after inoculation. Internal vascular discoloration was observed and the pathogen was reisolated from the symptomatic seedlings. No symptoms developed on any of the 10 control seedlings. On the basis of morphology and DNA sequence data, this pathogen is identified as F. oxysporum f. sp. canariensis. Fusarium wilt of Canary Island date palm has been previously reported from California, Florida, Nevada, Texas, and South Carolina (1). To our knowledge, this is the first report of Fusarium wilt of Canary Island date palm caused by F. oxysporum f. sp. canariensis in Louisiana, extending its geographic range. The disease may adversely affect the tradition of planting Canary Island date palms in New Orleans. The sequence of isolate PDC-4701 has been submitted to the NCBI database (GenBank Accession No. JF826442) and a culture specimen has been deposited in the Fusarium Research Center culture collection (Accession No. O-2602) at the Pennsylvania State University, University Park, PA. References: (1) M. L. Elliott et al. Plant Dis. 95:356, 2011. (2) D. M. Geiser et al. Eur. J. Plant Pathol. 110:473, 2004.