Host Raising of Broccoli, Brassica oleracea Italica, in Pot 

 

Fifteen days old broccoli seedlings and pot mixture were moved to the Horticulture Section, School of Agriculture and Food Sciences (SAFS), University of Queensland, Gatton on July 8, 2014. Pot mixture transferred to the 50 pots and a single broccoli seedling in each pot. Pots were then transferred to Bay-8 inside the glasshouse and were kept at a distance of about 35 to 45 cm from each other.
After transferring pots into the glasshouse, water was given to the young transferred broccoli seedlings through hand held hose. Broccolis were then observed regularly and water was provided as per plant requirement. These potted broccoli were then used for mass production of P. xylostella in insectary. 

 

Rearing of P. xylostella in the laboratory  

 

Stock population of P. xylostella larvae used in this study pupae were collected from broccoli (Brassica oleracea var Italica) crop in University of Queensland, Gatton Campus. To maintain stock culture of P. xylostella, pupae collected from the research field and transferred to rearing cages in insect rearing room on different potted broccoli plants already raised in a controlled environment of the glasshouse. Rearing was maintained in a controlled room at photoperiod of 16:08 (light: dark) hours, temperature 25±2°C and 55±07% relative humidity in the insect rearing room by using the modified rearing procedure of [21]. Pupae were held in lab in wooden-framed net cages (45 x 45 x 45 cm3) with cotton swabs containing a 10% sugar solution were available for the emerging adults and broccoli seedling for egg laying. They were kept caged until adults emerged and mated.  

 

After females oviposited, seedlings with eggs were removed from the cages and placed into different cages until the eggs hatched. The seedlings with newly-hatched larvae were transferred to big cages (120 x 60 x 60 cm3) in the insect rearing room where they were kept until the larvae pupated. Pupae were then harvested and transferred to the laboratory, some for mating immediately, the rest to be kept at 4˚C until needed to maintain the population. 

 

Plant Material  

 

For the research, neem, Azadirachta indica insecticidal chemicals in 0.31, 0.50, 0.60 and 1% extracts dilutions were provided by ‘BioAust’ Stafford Heights Queensland 4053. 

 

Effect on larval and pupal weight (mg) 

 

To determine the effect of neem extracts on growth inhibition of P. xylostella larvae, ten third instars larvae in each treatment were exposed using the leaf dip test and the ultimate effect on larval and pupal weights (mg) were observed.  

 

Effect on oviposition and hatching  

 

From 60 days old broccoli seedling, leaves were detached and dipped into one of five different experimental concentrations. Each impregnated leaf was put in the small container which held some water to maintain the moisture. The detached leaves were put in a container (3 cm in diameter, 11 cm in height) together with a cotton swab moistened with 10% of honey solution. Two female and two male adults  (3-4 days old) were released in the container and each treatment was replicated three times, giving a total of 60 adults used for these experiments. The total numbers of eggs deposited on the leaves were counted after 3 days and then the numbers of hatched eggs (F1) were recorded after 3 days. 

 

Antifeedant Effect (Larval Stage) 

 

Treated leaf discs (3 cm in diameter) with various concentrations were placed on a tray and left in a fume cabinet for 2 hours to evaporate any remaining solvents. For each treatment there were four leaf disc replicates; a total 12 same age larvae were used in each treatment. After being starved for 4 hours, the third instars of P. xylostella were exposed to the extracts impregnated leaf discs with choice and no-choice options. For the choice method, 3 larvae of the third instar were placed on treated and untreated/control leaf discs which were arranged around a plastic container, while for the no-choice method, the treated leaf discs and controls were placed in separate plastic containers (9 cm dia; 6cm height). After 24 hours, larvae were removed and faces brushed from the leaf discs, then the leftover leaf discs surfaces were placed on a piece of paper for digitizing the form of the leaf disc by using an Epson scanner at 300 dpi. After that, damage areas were measured using a computer programme, Image 1.41 (open sources program on http://rsb.info.nih.gov/ij). The antifeedant index (AFI) for the choice method was calculated by means of the equations (C-T)/(C+T) * 100%, and for the no- choice method (C-T)/C *100%, where C and T (mm2) denoted the consumed area of control and treated discs respectively [22].  

 

Data Analysis 

 

All the replicated data were statistically analyzed by using analysis of variance (ANOVA) technique appropriate for completely randomized design (CRD) [23] by using a statistical software “Statistics 8.1®” version. The significant means were separated by Fischer’s protected least significant difference test (LSD) after a significant F-test at α 0.05 level of probability. 

Effect on larval and pupal weight 

 

A dose dependent decrease was observed in larval and pupal weights (Table-1). In general, as the concentration of neem extracts increased, larval and pupal weight decreased. For the larval stage, there was about 56.43-68.20% reduction in weight caused by 0.31, 0.5, 0.6 and 1% concentrations. For the pupal stage, the weights were reduced by about 24.47-46.77%. The decrease in larval and pupal weight was significant among the different neem concentrations.  

 

All concentrations showed weight reductions both in larval and pupal stages of P. xylostella. According to [24] plant oils of A. calamus caused growth and feeding inhibition in S. litura. This was found to be due to the component β-asarone which acts as an antifeedant and growth inhibitor [24-26].  

 

Effects of various neem extracts on P. xylostella larvae and pupal weight have been well documented [27].Generally, azadirachtin extracts or azadirachtin-based insecticides are effective against P. xylostella larvae with significant lethal and antifeedant effects accompanied by significant reduction in food consumption and ultimately on their weight. These azadirachtin concentrations were toxic to all larval instars and pupae. A different economic threshold for the major lepidopterous pests may be needed when azadirachtin-based or other bioinsecticides are the primary components in the management programs. The effects on different developmental stages of P. xylostella were not always significantly different, although their azadirachtin contents vary greatly: 0.15% in Agroneem, 0.25% in Neemix, and 3% in Ecozin. Nevertheless, it has been proved that biological activity of azadirachtin-based insecticides cannot be solely judged by the azadirachtin content and azadirachtin extracts can contain numerous other chemicals, i.e. aflatoxins, salannin, nimbandiol, etc. that also have insecticidal activities and cause a great reduction in larval and pupal weight [28]. In addition, differences in extraction process, formulation solvent and adjuvant can dramatically influence toxicity and biological activity [28,29]. 

 

Effect of oviposition and hatching 

 

Increasing concentrations of neem oil decreased egg production and subsequent larval emergence as adults which developed from treated larvae. At all concentrations tested, mean egg laying was reduced significantly from 53.75 to 200.25 when compared to the control and mean egg hatch was significantly reduced from 271.75 in the controls to 18.75 in treated samples (Table-2). 

 

There are few published results available on the reproduction-inhibiting effects of L. petersonii oil on P. xylostella. The chemical components responsible for this activity have not been investigated further. A group of chemicals likely to be involved in this activity include neral, which was reported as being an oviposition deterrent effect on the leafhopper, Amrasca devastans (Distant.) (Hemiptera: Cicadellidae) [30]. 
The neem oil had potential insecticidal action since it was observed in this study that at 1%, this oil caused more than 50% mortality of P. xylostella. It produced strong antifeedant effects and insect growth inhibition in P. xylostella. In addition, the adult survivors produced less egg than the control insects. 

 

Other researchers also concluded that compounds in the neem extracts other than azadirachtin might be responsible for oviposition deterrent effects [28, 31, 32]. Suggested that non-volatile neem components contain primarily azadirachtin, appeared to be associated with azadirachtin concentration. 

 

Neem extracts and Neem-based insecticides appear to significantly deter oviposition many lepidopterous pests [33, 34, 35, 36] found that neem extracts reduced P. xylostella oviposition[37] found that the azadirachtin extract Margosan-O exerted a significant inhibitory effect on the oviposition of P. xylostella. Nonetheless, [38] reported that cabbage plants treated with neem seed kernel water extracts were more attractive for egg-laying by P. xylostella both in the laboratory and in the field. The possible explanation may be that P. xylostella moths were attracted to cabbage leaves by their stronger volatile that overshadows the odor of neem and deposited more eggs. 
Antifeedant index choice and no choice 

 

Maximum Anti Feeding Index (AFI) was recorded for 1% concentration whilst the lower was observed for 0.31% for 3rd instar larvae in choice experiment and same situation were noticed for 4th instar larvae (Table-3). 

 

In the no choice experiment, maximum area of the untreated leaf was consumed by the 3rd instar larvae followed by leafs treated with neem concentrations 0.31, 0.5, 0.6 and 1% which were 0.12, 0.08, 0.01 and 0.00 cm respectively (Table 4). High AFI were recorded for 1 and 0.6% concentration whilst the lowest was for 0.31% in the experiment. Likewise, 4th instar larvae also behave in the same way like 3rd instar larvae in the no choice experiment after 24 hours exposure to the leaves treated with different neem concentrations. Maximum areas of the leaves were consumed of untreated leaves whilst lowest areas of the leaves were consumed treated with 0.31% concentration. AFI for the 4th instar larvae showed that no significant of different neem concentrations effect has been noticed on 4th instar larvae. 

 

An antifeedant effect on larvae of the diamondback moth, P. xylostella was observed on Agroneem, Ecozin and Neemix treated cabbage, Brassica oleracea leaves and larvae fed on neem-based insecticide-treated leaves were smaller than those fed on non-treated controls [20, 39] reported that aqueous plant extract against P. brassicae, at different concentrations caused 3.2 to 81.8 percent protection to the foliage over control [39]. Extract of A. indica resulted in maximum protection to foliage against P. brassicae. Similarly,[40] reported that 2% leaf water extract of A. indica had a significant antifeedant effect, thereby reducing mean cabbage leaf area consumption of P. brassicae. However, [41] reported feeding inhibition up to 90 percent against the third instar larvae of Leptinotarsa decemlineata with crude leaf extract of A. indica. [42] Also reported feeding deterrent activity of aqueous extract of M. azedarach and A. indica against the larvae of P. xylostella. The strategy that can include antifeedants is the stimulodeterrent diversionary strategy (SDDS), sometimes called the “push-pull” strategy [43]. Further work is however required to fully understand its mode of action. It is now accepted that neem have a wide margin of safety for both user and consumer. Antifeedant effects of neem products have been reported to persist after replacement of treated leaves with untreated ones’ and similar effects were noted in the present work. These observations, together with evidence from topical application and injection studies, suggest that neem products may possess anorectic activity.  

 

A reduced rate of feeding in the presence of neem extracts may also explain the time taken (48-120 h) to accumulate a dose lethal to the majority of the population. Some studies have reported habituation as a reason to feed but have not been able to distinguish insects that accept treated food or the ability of insects to reduce the chemical responsible for their aversive behavior more readily because they were hungry [44] based on this reason, it suggests that habituation might occur during this stage. Habituation or the ability of insect to reduce their aversive behavioral response to unpleasant or toxic compounds in a plant can be recorded if the insect is repeatedly exposed to toxic plant over a period of time [45]. 

 

It is concluded that, these concentrations should enhance the management of lepidopterous pests in the vegetable agroecosystems because they do not persist in the environment, have unique modes of action, low mammalian toxicity, and may be potentially compatible with natural enemies. Neem extracts seem to be suitable for combination with biological control, as these extracts have a profound effect on larval pupal weight, oviposition and hatching and also on antifeedant effect. 

 

Concentrations (%)	Larval wt ±SE (mg)	Larval wt decrease over control (%)	Pupal wt ±SE (mg)	Pupal wt decrease over control (%)
0	3.91±0.04 a	---	6.30±0.09  a	---
0.31	1.70±0.14  b	56.43	4.76±0.13  b	24.47
0.5	1.44±0.02  c	63.28	4.41±0.13  c	29.99
0.6	1.40±0.07  c	64.24	4.18±0.02  c	33.76
1	1.24±0.04  c	68.20	3.36±0.09  d	46.77
LSD value (0.05 )	0.23	---	0.31	---

 

 

 

 

 

 

 

 

 

 

 

 

For each larval and pupal weight means with sd (standard deviation) with the same letter are not   significantly different (Least Significant Difference test, p<0.05)

 

Table 1:  Effect of different Neem Concentrations on Larval and pupal Weights (milligram) of P. xylostella (L.)

 

Concentrations (%)	Mean # Eggs	Mean eggs hatched
0	272.50 a	271.75 a
0.31	200.25 b	153.25 b
0.5	160.25 c	105.50 c
0.6	97.50 d	70.75 d e
1	53.75 e	18.75  d
LSD value (0.05 )	22.87	19.24

 

Means in columns followed by different letters are significantly different at α = 0.05 level of significance by using Fischer's Protected LSD test.

 

Table 2: Effect of different Neem Concentrations on oviposition and hatching of P. xylostella (L.)

 

Concentrations (%)	Area consumed by  3rd instar larvae (T) (cm)	Area consumed in (C) (cm)	AFI for 3rd instar	Area consumed by 4th instar larvae (T) (cm)	Area consumed in (C) (cm)	AFI for 4th instar
0.31	0.42	1.95	63.39 b	0.28	2.87	82.34 b
0.5	0.12	2.73	91.73 a	0.30	2.78	80.52 b
0.6	0.03	2.78	97.86 a	0.15	2.84	90.29 ab
1	0.00	2.55	99.80 a	0.00	2.95	99.66 a
LSD value (0.05 )	---	---	18.47	---	---	16.97

 

 

Means in columns followed by different letters are significantly different at α = 0.05 level of significance by using Fischer's Protected LSD test.

 

Table 3: Antifeedant Index (AFI) for 3rd and 4th instar larvae of P. xylostella after 24 hours exposure to different neem concentrations treated leaves (Choice Method).

 

Concentrations (%)	Area consumed by 3rd instar larvae (T) (cm)	AFI for 3rd instar larvae	Area consumed by 4th instar larvae (T) (cm)	AFI for 4th instar larvae
0.31	0.12	96.01b	0.1	96.42 a
0.5	0.08	97.40 ab	0.15	94.94 a
0.6	0.01	99.74 a	0.23	92.06 a
1	0	99.91 a	0	100.00 a
Control	2.88	---	2.87	---
LSD value (0.05 )	---	2.73	---	9.19

 

 

Means in columns followed by different letters are significantly different at α = 0.05 level of significance by using Fischer's Protected LSD test.

 

Table 4: Antifeedant Index (AFI) for 3rd and 4th instar larvae of P. xylostella after 24 hours exposure to different neem concentrations treated leaves  (No Choice Method).